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STUDIES  IN  PLANT  RESPIRATION  AND 
PHOTOSYNTHESIS 


BY 

Hf  AV^SroEHR  AND  J.  M.  McGee 


Published  by  the  Carnegie  Institution  op  Washington 
Washington,  February.  1923 


®{]E  p,  ^.  ^iU  pbrarg 


^ 


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C^V9 


CARNEGIE  INSTITUTION  OF  WASHINGTON 
Publication  No.  325 


JUDD    &    DETWEILER,    INC., 
WASHINGTON^  D.  C. 


N.C. 


<r^-v'' 


PREFACE. 


The  experiments  on  photosynthesis  which  were  begun  at  the 
Desert  Laboratorj'-  in  Tucson,  Arizona,  about  eight  years  ago 
yielded,  among  other  things,  the  conclusion  that  prerequisite  to  an 
understanding  of  the  nature  of  the  energy  transfer  in  photosynthesis 
was  a  more  extensive  knowledge  of  the  metabolism  of  chlorophyllous 
organs.  It  became  clear  that,  before  attempting  to  follow  the 
course  of  the  synthesis  of  carbohydrate  material  in  illuminated 
leaves,  it  was  essential  to  know  more  about  the  conditions  governing 
the  equilibria  and  mutual  transformations  of  the  various  groups  of 
carbohydrates,  quite  independent  of  the  photosynthetic  process. 

In  publication  No.  287  some  of  the  more  essential  of  these  con- 
ditions were  described.  A  further  prerequisite  to  an  understanding 
of  the  carbohydrate  economy  of  chlorophyllous  leaves  is  more 
precise  information  regarding  the  nature  of  the  carbohydrate  catabo- 
lism  and  the  conditions  governing  this  phenomenon.  In  the  present 
publication  are  described  the  results  of  experiments  on  the  relation 
of  the  amino-acid  and  carbohydrate  content  to  the  respiration  of 
leaves.  The  information  gained  from  these  studies,  as  well  as  from 
those  of  a  number  of  other  workers  in  this  field,  helps  to  emphasize 
the  fact  that  photosynthesis  is  an  exceedingly  complex  process. 
The  present  status  of  the  problem  is,  however,  exceedingly  encourag- 
ing in  that  refinement  of  methods  of  observation  and  experimentation 
and  the  utilization  of  the  results  and  conceptions  of  allied  physical 
sciences  are  contributing  greatly  to  a  better  understanding  of  the 
phenomenon  of  photosynthesis. 

This  publication  comprises  the  results  of  investigations  carried 
out  during  1919-1922.  It  is  a  pleasure  to  acknowledge  here  the 
assistance  rendered  in  the  preliminary  part  of  this  work  by  Dr. 
Frances  Long. 

H.  A.  Spoehr. 
Coastal  Laboratory, 

Carmel,  California,  June,  1922. 


:^-w 


CONTENTS. 

Part  I.  The  Carbohydrate-Amino-Acid  Relation  in  the  Respiration  of  Leaves. 

Introductory  discussion 1 

Methods  and  apparatus 21 

1.  The  experimental  material 21 

2.  The  apparatus 23 

3.  The  procedure  of  the  respiration  experiments 30 

4.  The  analyses 33 

Experimental  results 35 

1.  The  normal  course  of  respiration 35 

2.  Glycocoll 43 

3.  d-glucose . . 45 

4.  Sucrose 55 

5.  d-levulose 57 

6.  d-mannose 64 

7.  Effect  of  the  natural  increase  in  amino-acids.     Influence  of  light  on  amino-acids 
and  effect  on  respiration 66 

8.  The  action  of  amino-acids  on  sugars 75 

Part  II.  The  Internal  Factor  in  Photosynthesis. 

Introductory  discussion •. 76 

Methods  and  apparatus 84 

1.  The  experimental  material 84 

2.  The  apparatus ^  85 

Experimental  results 90 


STUDIES  IN  PLANT  RESPIRATION  AND 
PHOTOSYNTHESIS. 


Bt  H.  a.  Spoehr  and  J.  M.  McGee. 


I.   THE  CARBOHYDRATE-AMINO-ACID  RELATION  IN 
THE  RESPIRATION  OF  LEAVES. 
INTRODUCTORY  DISCUSSION. 

Already  at  the  beginning  of  the  nineteenth  century  it  had  been 
realized  that  the  materials  which  support  the  respiratory  activity 
in  plants  are  carbohydrates  and  fats.  The  observations  of  Lavoisier 
and  of  De  Saussure  that  the  respiration  coefficient,  CO2/O2,  in 
mature  higher  plants  is  usually  very  close  to  unity  were  even  at  that 
time  accepted  as  evidence  of  the  preponderance  of  carbohydrates 
as  the  fuel  material.  Since  then  a  continually  increasing  number 
of  chemical  compounds  has  been  discovered  in  plants.  When 
derived  from  the  higher,  autotrophic  plants,  these  multifarious 
products  must  be  considered  as  coming  primarily  from  the  carbohy- 
drates through  the  various  intricate  channels  of  plant  metabolism. 
So  while  the  substances  which  are  used  as  sources  of  energy  by  the 
lower  plants  and  bacteria  are  of  great  variety,  the  higher,  mature 
chlorophyllous  plants  are  in  general  consumers  of  carbohydrates. 

It  would,  however,  be  drawing  a  very  incomplete  picture  of  plant 
respiration  if  this  were  confined  to  the  carbohydrates  and  fats  and 
the  more  elusive  and  complex  nitrogenous  components  were  to  be 
omitted.  But  unfortunately  our  knowledge  of  the  function  of 
nitrogen  compounds  in  the  metaboUsm,  particularly  of  the  higher 
plants,  is  most  fragmentary.  It  is  our  belief  that  a  rational  con- 
ception of  the  function  of  nitrogen  in  the  economy  of  the  higher 
plants  can  be  gained  only  through  an  analytic  study  of  definite 
groups  of  nitrogen  compounds.  This  belief  has  its  foundation  first 
of  all  in  the  fact  that  nitrogen  is  capable  of  forming  such  a  variety 
of  compounds  of  basically  different  physiological  properties,  and 
furthermore  because  our  chemical  knowledge  of  the  nitrogen  com- 
pounds derived  from  protoplasmic  proteins  is  fairly  well  organized 
and  related  and  so  can  serve  as  a  most  valuable  experimental  guide. 

Even  a  cursory  study  of  the  carbohydrate  economy  of  the  higher 
plants  must  emphasize  the  old  conclusion  that  physiology  is  essen- 
tially a  study  of  dynamics.  Thus  far  organic  chemistry  has  dealt 
largely  with  the  properties  and  reactions  of  carbon  compounds. 
The  recent  tendency  to  enter  into  various  phases  of  energetics  and  the 
fundamental  causes  of  chemical  reactivity  will  make  chemistry  an 
even  more  valuable  adjunct  to  physiology  than  heretofore. 

1 


2  STUDIES  IN  PLANT  EESPIRATION  AND  PHOTOSYNTHESIS. 

It  would  seem,  then,  that  the  true  province  of  plant  chemistry 
should  be  the  unraveling  of  the  tangle  of  chemical  reactions  which 
give  rise  to  this  mass  of  carbon  compounds  and  ultimately  to  deter- 
mine the  function  of  these  reactions  in  the  economy  of  the  living 
plant.  Historically,  what  is  now  termed  organic  chemistry  had  its 
origin  in  such  a  task.  But  the  chemist  discovered  that  he  could  find 
a  large  number  of  short  cuts;  that  the  intermediary  action  of  living 
things  was  not  essential  to  the  production  of  most  of  the  substances 
found  in  nature;  that,  in  fact,  he  could  greatly  improve  on  nature  in 
the  production  of  the  multifarious  substances  of  use  in  industry 
and  the  arts. 

In  some  respects  the  present  has  its  points  of  similarity  with  the 
time  of  Woehler  and  Liebig.  Much  sober  thought  is  now  being 
given  to  the  fundamental  difference  between  the  living  and  the 
non-living.  The  first  flush  of  success  of  the  purely  mechanistic 
interpretation  of  life  processes  has  paled  as  the  enormous  difficulties 
of  applying  this  conception  to  the  highly  intricate  and  manifold 
manifestations  of  life  have  been  more  clearly  recognized.  The 
methods  and  modes  of  transforming  matter  followed  by  the  chemist 
and  by  the  living  organism  are  perhaps  not  as  similar  as  appears  at 
first  glance.  At  first  we  were  primarily  interested  in  the  final  goal, 
the  products  of  material  transformations;  but,  in  chemistry  as  well 
as  in  physiology,  as  we  follow  more  closely  the  energetics  of  such 
transformation,  many  almost  irreconcilable  differences  arise  which, 
before  the  two  can  be  brought  together,  necessitate  sweeping  com- 
promises to  the  unknown  and  indefinite.  And  it  is  this  question  of 
energy  which  is  forcing  us  to  a  deeper  study  of  living  things.  The 
scientific  world  is  awakening  to  the  necessity  of  taking  stock  of  our 
available  sources  of  energy.  Repeatedly  attention  has  been  called 
to  the  inexhaustible  floods  of  solar  energy  and  to  the  extensive  use 
of  plant  products  to  drive  the  machinery  of  our  civilization.  Car- 
bohydrates are  to  prevent  the  too-rapid  exhaustion  of  our  oil  and 
coal  resources.  As  the  realization  of  the  limits  of  our  energy  re- 
sources has  become  more  general,  reliance  on  the  magic  accomplish- 
ments of  the  chemist  has  increased,  and  the  chemists,  with  cool 
theoretical  nonchalance,  have  pointed  to  the  untold  possibilities  of 
solar  energy.  But  the  living  chlorophyllous  plant  still  remains  the 
only  converter  of  solar  energy! 

The  chemical  reaction  most  fundamental  to  all  living  things, 
the  photosynthetic  process,  the  bridge  between  the  inanimate  and 
the  animate,  the  source  of  most  of  our  energy,  has  stubbornly  resisted 
all  attempts  at  solution  by  physico-chemical  methods.  Here  it  has 
become  plainly  evident  that  while  physiology  is  rife  with  chemical 
possibilities,  physiological  experience  alone  is  the  guide  to  the  inter- 
pretation of  biological  processes.     Furthermore,  it  has  become  clear 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS.  6 

that  physiology  is  a  great  deal  more  than  applied  physics  and  chem- 
istry, although  we  must  rely  upon  these  discipUnes  in  order  to  form 
conceptions  of  the  various  vital  phenomena  as  operations  of  known 
causes.  With  complete  disregard  of  biological  facts,  chemists  have 
continued  to  evolve  theories  of  the  chemistry  of  photosynthesis 
and  to  supplement  and  modify  existing  theories.  The  fundamental 
fallacy  in  these  speculations  has  been  that  photosynthesis  has  been 
regarded — to  quote  a  recent  technical  article — as  ''simply  a  manu- 
facture that  provides  material  used  in  the  process  of  living,  "^  that 
because  a  process  can  be  suspended  for  hours  and  months  it  is  not  a 
process  or  function  of  the  Uving.  The  many  attempts  which  have 
been  made  to  reproduce  the  photosynthetic  process  apart  from  the 
Uving  cell  have  not  been  successful.^  This  also  appHes  to  the  experi- 
ments in  which  various  chlorophyll  preparations  have  been  used, 
as  well  as  to  those  in  which  ultra-violet  Ught  was  employed  as  a 
source  of  radiant  energy,  although  the  latter  conditions  have  no 
direct  bearing  on  photosynthesis,  because  normally  the  plant  makes 
use  of  only  the  radiations  in  the  visible  spectrum. 

That  the  photosynthetic  process  is  intimately  associated  with 
the  protoplasmic  activity  of  the  hving  cell  was  recognized  a  long  time 
ago.  Boussingault,^  in  his  studies  of  the  effect  of  gases  such  as 
hydrogen,  nitrogen,  and  methane  on  photosynthesis,  came  to  this 
conclusion.  The  same  conclusion  was  reached  and  somewhat 
elaborated  by  Pringsheim."  Interesting  and  valuable  as  these 
older  investigations  are,  as  well  as  the  later  ones  of  Ewart,^  Httle 
precise  information  can  be  gained  from  them,  because  the  methods 
of  experimentation  and  observation  are  naturally  not  in  accord  with 
present  standards  of  accuracy. 

It  has  become  evident  that  further  insight  into  the  complex 
phenomenon  of  photosynthesis  is  to  be  gained  only  by  means  of 
intensive  study  of  the  process  under  very  carefully  controlled  external 
conditions  and  the  ehmination  of  secondary  factors.  Unfortunately, 
however,  in  the  study  of  photosynthesis,  internal  conditions,  such 
as  available  nutritional  material,  have  been  totally  neglected  and 
httle  or  no  regard  has  been  paid  to  the  previous  history  of  the  plant. 
It  is  highly  probable  that  this  neglect  in  many  instances  accounts 

1  Scientific  American  Supplement,  No.  2257,  223  (1918). 

*  WiLLSTAETTER,  R.,  and  A.  Stoll.     Untersuchungen  ueber  die  Assimilation  der  Kohlensaeure. 

1918,  391-415. 
Spoehh,  H.  a.     The  theories  of  photosynthesis  in  the  light  of  some  new  facts.     Plant  World, 
19,  1-16  (1916). 
'  BoussiNGAULT,  J.  B.     ^Itude  sur  les  fonctions  des  feuilles.    Compt.  rend.,  60,  608;  1865,  Agro- 
nomie,  4,  359-397  (1868). 

*  Pbingsheim,  N.     Ueber  die  Abhaenigkeit  der  Assimilation  Gruener  Zellen  von  ihrer  Sauer- 

stoffathmung,  und  den  Ort,  wo  der  im  Assimilationsacte  der  Pflanzenzelle  gebildeter  Sauer- 
stoff  entsteht.     Sitzber.  Preus.  Akad.  Wiss.,  763-777  (1887). 
'  EwART,  A.  J.     On  assimilatory  inhibition  in  chlorophyllous  plants.     Jour.  Linnean  Soc,  31, 
364-^61  (1896);  556  (1897). 


4  STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 

for  the  contradictory  observations  and  reports  which  have  been 
made  by  different  investigators  on  various  phases  of  the  phenomenon 
of  photosynthesis.  Furthermore,  wherever  consideration  must  be 
given  to  the  respiratory  activity  of  plants,  it  is  essential  that  all 
possible  information  be  obtained  regarding  the  internal  conditions, 
as,  for  instance,  available  carbohydrate  supply.  Such  data  can  be 
obtained  only  by  rather  laborious  analytical  methods. 

If  such  an  interdependence  between  photosynthesis  and  respiration 
actually  exists,  a  better  understanding  of  the  nature  of  respiratory 
activity  is  an  absolute  prerequisite  to  further  investigation  of  this 
relationship. 

WMle  any  consideration  of  the  relation  of  photosynthesis  to 
respiration  must  take  into  account  the  carbohydrate  economy  of 
the  plant,  it  is  evident  that  respiratory  activity  itself  can  not  be 
interpreted  solely  on  the  basis  of  carbohydrate  balance  and  supply. 
In  fact,  it  has  been  known  for  some  time  that  there  is  no  direct 
relation  between  the  amount  of  available  carbohydrates  and  the 
rate  at  which  the  organism  uses  this  material  in  its  respiratory 
activity.  Although  carbohydrates  constitute  the  major  portion  of 
the  material  used  by  the  plant  in  its  process  of  respiratory  com- 
bustion, the  other  agencies  and  factors  which  play  a  role  in  this 
process  are  still  but  vaguely  known.  Our  knowledge  of  the  chemical 
possibihties  which  would  find  application  here  is  still  very  incomplete. 

Plant  physiologists  have  generally  accepted  the  dictum  that, 
given  an  adequate  supply  of  carbohydrates,  water,  certain  inorganic 
salts,  and  the  proper  temperature,  the  rate  of  life  processes  in  the 
higher  plants  depends  upon  the  active  mass  of  protoplasm.  To  the 
activity  of  protoplasm  have  been  ascribed  all  those  functions  and 
reactions  which  could  not  be  described  in  the  terms  and  through 
existing  conceptions  of  physics  and  chemistry.  The  attempts  of 
MacDougal,  Loeb,  and  others  to  describe  the  physical  behavior  of 
protoplasm  on  the  basis  of  colloidal  phenomena  have  done  much 
to  break  away  from  this  tendency  and  to  determine  to  what  extent 
protoplasmic  activity  is  amenable  to  physical  simulation.  Simi- 
larly, attempts  have  been  made  by  Palladin  to  correlate  certain 
components  of  the  protoplasm  with  the  respiratory  activity  of  the 
plants. 

The  carbohydrate-nitrogen  relation  has  of  late  found  appHcation 
in  the  investigations  of  Kraus  and  Kraybill,i  who  have  formulated 

'Kraus,  E.  S.,  and  H.  R.  Kraybill.     Vegetation  and  reproduction  with  special  reference  to 

the  tomato.     Oregon  Agr.  Exp.  Sta.  Bull.  149  (1918). 
Kraus,  E.  S.     The  modification  of  vegetative  and  reproductive  functions  under  some  varying 

conditions  of  metabolism.     Amer.  Jour,  of  Bot.,  7,  400-416  (1920). 
Harvey,  E.  M.,  and  A.  E.  Murneck.     The  relations  of  carbohydrates  and  nitrogen  to  the 

behavior  of  apple  spurs.     Oregon  Agr.  Exp.  Sta.  Bull.  176  (1921). 
Hooker,  H.  D.,  Jr.     Seasonal  changes  in  the  chemical  composition  of  apple  spurs.     Misaouri 

Agr.  Exp.  Sta.  Research  Bull.  40  (1920). 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS.  5 

a  general  theory  which  has  met  widespread  success  in  its  practical 
bearing  on  agriculture.  Their  investigations  deal  in  the  main  with 
the  relation  between  vegetation  and  fruit  setting  and  nitrate  fer- 
tiHzation.  They  have  been  able  to  differentiate  the  carbohydrate- 
nitrogen  relations  of  certain  plants  on  the  basis  of  their  vegetative 
and  sexual  activities. 

There  exists  very  httle  exact  information  regarding  the  general 
course  of  formation,  synthesis,  or  fate  in  the  general  metabolism 
of  any  of  the  organic  nitrogen  compounds  in  higher  plants.  The 
number  and  variety  of  different  types  of  nitrogenous  substances 
found  in  plants  are  enormous,  and  it  appears  an  almost  hopeless 
task  to  unravel  the  tangle  of  chemical  reactions  which  lead  to  this 
multiplicit}^  of  compounds.  It  seems,  however,  quite  erroneous  to 
class  together  the  various  types  of  nitrogen  compounds,  such  as  the 
proteins  and  related  substances,  the  amino-acids,  amides,  etc.,  and 
regard  their  phj'^siological  functions  as  being  the  same  or  even  very 
intimately  connected.  An  insight  into  this  complex  of  chemical 
reactions  comprising  metabolic  energesis  can,  of  course,  be  obtained 
only  through  laborious  experimental  investigation  involving  much 
observational  and  chemical-analytical  work  with  living  plants. 
Chemistry  can  offer  no  short  cuts,  but  rather  supplies  the  instruments 
with  which  to  illuminate  these  hidden  activities.  Nor  can  the 
conceptions  of  chemistry  always  find  immediate  application  to  reac- 
tions in  living  organisms.  Thus  the  many  theoretical  speculations 
regarding  the  chemistry  of  the  photosynthetic  process  have  contrib- 
uted very  little  to  the  solution  of  that  problem.  This  is  true  also 
of  the  chemical  speculations  regarding  the  formation  of  the  amino 
acids  and  the  proteins,  although  there  are  a  number  of  elaborate 
theories  which  have  been  formulated  to  explain  this  complex  process.^ 
To  these  the  warning  of  Pfeffer  applies  very  aptly,  which  is  to  the 
effect  that  it  is  a  very  confusing  error  to  presume  that  an  organism 
must,  in  its  metabolic  economy,  follow  a  course  which  seems  to  man, 
under  the  influence  of  existing  chemical  and  physical  knowledge, 
the  most  plausible  course.  All  of  these  theories  are  still  dealing  with 
probabilities  supported  by  very  little  experimental  physiological 
evidence.  Moreover,  plant  chemistry  can  hardly  claim  to  be  an 
independent  science.  The  great  advances  which  have  been  made  in 
protein  chemistry  have  been  stimulated  largely  by  animal  physiology, 
and  progress  in  our  understanding  of  plant  proteins  has  been  made, 
in  the  main,  by  following  the  methods  previously  worked  out  for 
proteins  of  animal  origin.     These  studies  seem  to  indicate  that  the 

1  LoEB,  W.     Ber.  d.  deutsch.  chem.  Ges.,  46,  684-697  (1913). 
Franzen,  H.     Jour.  Prakt.  Chem.,  86,  133  (1913). 

Batjdish,  O.     Jour.  Biol.  Chem.,  48,  489-502  (1921).     Ber.  d.  deutsdi.  chem.  Gps.,  46,  115-125 
(1913);  49,  1159-1167  (1916);  50,  652-660  (1917);  51,  793-805  (1918);  52,  B  35-43  (1919). 
LoEW,  O.     Ibid.,  SO,  909-910  (1917). 


6  STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 

proteins  of  plant  and  animal  origin  are  very  similar  in  composition. 
It  is,  nevertheless,  an  open  question  as  to  what  degree  we  are  justified, 
on  the  basis  of  this  analogy,  in  drawing  conclusions  as  to  the  function 
of  the  proteins  in  the  two  types  of  organisms. 

The  extraction  of  proteins  from  leaves  is  associated  with  consider- 
able difficulty.  Winterstein^  demonstrated  this  by  the  use  of  dif- 
ferent methods  on  a  variety  of  plants.  These  difficulties  arise  from 
the  fact  that  the  proteinaceous  material  is  quite  indiffusible  and 
clings  tenaciously  to  the  structural  elements  of  the  plant,  and  that 
any  methods  of  extraction  or  expression  yield  complex  mixtures 
from  which  the  protein  can  be  freed  only  by  means  of  a  variety  of 
substances  which  in  some  degree  also  affect  the  proteins.  Very 
little  systematic  work  has  been  done  on  the  proteins  with  a  view  to 
determining  which  are  present  in  green  leaves.  A  good  beginning 
has  been  made  by  Chibnall  and  Schryver,^  who  attempted  to  work 
out  methods  for  isolating  and  identifying  the  proteins  in  the  leaves 
of  some  of  the  higher  plants.  Osborne,  Wakeman,  and  Leavenworth^ 
have  also  outlined  a  procedure  for  the  separation  of  various  protein 
constituents  from  the  alfalfa  plant. 

Mature  leaves  are  relatively  high  in  proteins.  The  mesophyll, 
freed  from  leaf-veins,  often  shows  values  for  protein  of  over  30  per 
cent  of  the  dry  material.  Lakon,^  by  means  of  the  Molish  reaction, 
was  able  to  demonstrate  decided  differences  in  the  amount  of  protein 
in  variegated  leaves.  The  chlorophyllous  portions  gave  a  very 
intense  protein  reaction,  while  the  albescent  portions  were  poor  in 
proteins.  This  seems  to  confirm  the  opinion  that  in  leaves  the  prin- 
cipal masses  of  protein  are  located  in  or  about  the  chloroplasts. 
In  fact,  Meyer^  found  that  as  leaves  become  older  the  reduction  of 
the  green  color  runs  parallel  with  a  reduction  in  the  protein-content 
of  the  leaves.  According  to  these  investigations  the  leaf  proteins 
are  located  chiefly  in  the  chloroplasts,  while  the  nucleus  and  cyto- 
plasm seem  but  very  slightly  affected  by  variations  in  protein- 
content.  Thus  the  size  of  the  chloroplasts  is  greatly  influenced  by 
the  amount  of  protein  in  the  leaf,  in  that,  as  the  proteins  disappear 
when  the  leaf  is  kept  in  the  dark,  the  chloroplasts  become  smaller 
and  contain  less  chlorophyll,  while  with  an  increase  in  proteins  the 

>  WiNTERSTEiN,  E.     Ueber  die  Sticksto£fhaltigen  Bestandtheile  greuner  Blaetter.     Ber.  d.  deutsch. 

hot.  Ges.,  19,  326-330  (1901). 
Also  Hamilton,  T.  S..  W.  B.  Nevens,  and  H.  S.  Grindley.     The  quantitative  determination 

of  amino-acids  of  feeds.     Jour.  Biol.  Chem.,  48,  249-272  (1921). 
s  Chibnall,  A.  C,  and  S.  B.  Schryver.     Investigations  on  the  nitrogenous  metabolism  of 

higher  plants.     Biochem.  Jour.,  15,  60-75  (1921). 
»  Osborne,  T.  B.,  A.  J.  Wakeman,  and  C.  S.  Leavenworth.     The  proteins  of  the  alfalfa  plant. 

Jour.  Biol.  Chem.,  49,  63-91  (1921). 
*  Lakon,  G.     Biochem.  Zeitschr.,  78,  145-154  (1916). 
6  Meyer,   A.     Eiweisstoffwechsel   und  Vergilbung  der    Laubblaetter   von    Tropaeolum  majua. 

Flora,  11,  85-127  (1918). 
Cf.  also  CaAPEK,  F.     Biochemie  der  Pflamen,  2d  ed.,  3,  293.     Jena  (1920). 


STUDIES  IN  PLANT  RESPIEATION  AND  PHOTOSYNTHESIS.  7 

reverse  occurs.  In  this  connection  it  is  interesting  to  note  that 
according  to  the  experiments  of  Deleano^  it  is  claimed  that  when 
leaves  {Vitis  vinifera  at  18°  to  20°)  are  kept  in  the  dark,  these  util- 
ize only  carbohydrates  during  the  first  hundred  hours;  thereafter 
drastic  disturbances  in  the  proteinaceous  components  of  the  leaf 
occur.  The  carbohydrates  thus  act  in  the  nature  of  protein-sparers. 
Now,  Meyer  points  out  that  when  leaves  are  kept  in  the  dark  for  a 
long  time,  the  chloroplast  apparatus  becomes  so  greatly  impaired 
through  loss  of  protein  that,  on  subsequently  placing  the  leaves  in 
the  Hght,  photosynthesis  is  no  longer  sufficiently  active  to  produce 
enough  material  to  cover  the  nocturnal  loss  through  respiration. 
Meyer's  observation  of  decreasing  protein-content  with  advancing 
age  of  the  leaves  is  especially  interesting  when  compared  with  the 
studies  of  Nicolas,^  who  found  that  on  the  basis  of  respiration  coeffi- 
cients the  energy  release  in  young  leaves  is  considerably  higher  than 
in  old  ones. 

According  to  Emmerhng,  there  is  with  advancing  age  only  a 
reduction  of  amino  nitrogen  and  total  nitrogen  but  not  of  protein 
nitrogen.  This  latter  observation  is  in  even  closer  accord  with  the 
hypotheses  of  a  causal  connection  between  amino-acid  content  and 
respiration.  Also,  the  carbohydrate-content  shows  a  general  increase 
as  the  leaves  grow  older.^  These  facts  indicate  that  carbohydrate- 
content  alone  can  not  be  taken  as  an  index  of  the  rate  of  respiration ; 
nor,  obviously,  can  the  carbohydrate-content  serve  as  a  measure  of 
the  photosynthetic  activity. 

The  following  analyses  taken  from  Czapek  {Biochemie  der  PHanzen, 
1st  ed.,  Vol.  II,  p.  202)  indicate  the  amounts  of  protein  which  have 
been  found  in  leaves.  These  values  have,  however,  only  a  limited 
significance,  because  in  the  analysis  neither  the  age  of  the  leaves, 
their  history,  nor  the  environic  conditions  were  taken  into  consider- 
ation. 


Table  1. 


Plant. 

Water- 
content. 

Crude 
protein 
NX6.25. 

Plant. 

Water- 
content. 

Crude 
protein 
N  X6.25. 

Lactuca  sativa 

Plantago  major 

V.ct. 

81^44 
10.10 

p.ct. 
31.75 
2.65 
10.20 
33.06 
27.37 

Ilex  paraguayensis 

p.ct. 
9.41 

16^29 

V.ct. 

4.51 

6.28 
21.23 
38.77 

5.10 

Allium  porrum 

Cichorium  endiva 

Coffea  arabica 

Spinacia  oleracea 

Vicia  cracca.  . 

1  Deleano,  N.     Jahrh.  f.  wiss.  Bot.,  51,  541-592  (1912). 
»  Nicolas,  G.     Reo.  Gen.  Bot.,  30,  210-225  (1918). 
MiCHEi/-DuKAND,  E.    lUd.,  30,337-345,  377-382  (1918); 

251-268, 287-317  (1919). 
»  Emmerung,  E.     Landw.  Verauch.  Stat.,  34,  113  (1880). 


31, 10-27,  53-60,  143-156,   196-204, 


8 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


The  relation  of  protein  nitrogen  to  non-protein  nitrogen  can  be 
seen  from  Pigorini's*  results  of  the  analysis  of  Morus  leaves  (table  2). 

The  true  function  of  the  leaf  proteins  is  a  question  on  which  there 
exists  very  little  definite  information,  although  it  has  been  considered 
one  of  the  most  vital  problems  in  plant  physiology.  In  the  nitrogen 
economy  of  animals  it  is  apparent  that  a  portion  of  the  assimilated 
proteins  serves  to  replace  the  'Svear  and  tear"  on  the  protoplasmic 
machinery.  For  this  Rubner  introduced  the  conception  ''repair 
quota"  of  protein.  Also  a  "growth  quota"  of  protein  is  necessary 
to  supply  material  for  the  development  of  protoplasm  in  new  cells 
in  growing  organisms.  When  protein  is  available  in  such  amounts 
beyond  the  requirements  of  "repair  quota"  and  "growth  quota," 
it  can  be  converted  into  glucose  and  fatty  acids  and  thus  serve  as 
fuel,  very  much  as  though  these  substances  had  been  ingested  as 
food.  To  such  material  Rubner  gave  the  term  "dynamic  quota." 
Under  conditions  of  copious  protein-supply  the  animal  can  to  a  degree 
store  or  deposit  the  protein  in  the  tissue  cells.  It  seems  to  be  an 
undecided  question  whether  such  protein  becomes  a  part  of  the 
living  tissue  or  is  stored  by  the  cells  very  much  as  glycogen  is.     The 

Table  2. 


Morning. 

Evening. 

1 
Dry  material.       Fresh  material. 

Dry  material. 

Fresh  material. 

Total  N 

I 
p.  ct.                        p.  ct. 
2  445                      0  772 

p.ct. 

2.534 

2.368 

.166 

p.  ct. 

0.884 

.824 

.059 

Protein  N 

2.309                         .729 
.1105                       .043 

Non-protein  N 

animal  organism,  unlike  the  plant,  is  constantly  losing  a  certain 
(luantity  of  nitrogen,  and  it  appears  that  if  this  quantity  is  not  re- 
])laced  by  ingested  protein-food  the  proteinaceous  body-tissue  is 
drawn  upon. 

In  the  autotrophic  plant  the  condition  seems  to  be  quite  different. 
Here  also,  however,  it  is  first  of  all  essential  that,  in  the  discussion, 
well-defined  conditions  be  established  and  that  generaUzations  be 
not  made  too  inclusive.  This  has  been  an  unfortunate  feature  of  the 
contributions  to  protein  metaboUsm  in  plants.  It  should  hardly 
need  emphasis  that  results  with  the  fungi  may  not  find  immediate 
application  to  autotrophic  plants,  nor  that  the  behavior  of  germinat- 
ing seeds  corresponds  to  mature  chlorophyllous  leaves.  In  general, 
it  appears  as  though  plants  were  exceedingly  economical  with  their 

'  C:zAPKK,  F.     Biochemie  der  Pfianzen,  vol.  ii,  294;  2d  ed.,  1920. 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS.  9 

nitrogen  compounds.  There  are  no  nitrogenous  excreta,  and  the 
plant  possesses  the  power,  to  a  very  highly  developed  degree,  of 
resynthesizing  the  decomposition  products  of  proteins.  The  excep- 
tion to  these  statements  should  be  mentioned  at  once.  Thus  there 
are  the  denitrifying  actions  of  certain  bacteria  and  the  behavior  of 
the  lower  non-chlorophyllous  plants.  These  organisms  exhibit  a 
behavior  toward  the  proteins  which  seems  to  differ  in  many  funda- 
mental aspects  from  that  of  the  higher  plants.^ 

In  this  study  we  are  concerned  primarily  with  the  metabolism  of 
mature  autotrophic  leaves.  A  priori,  the  nitrogen  metabolism  of 
such  organs  would  seem  to  have  but  little  similarity  to  that  of  ger- 
minating seeds,  in  which  it  is  primarily  a  question  of  the  resorption 
of  stored  proteinaceous  material.  In  view  of  its  great  importance 
it  is  surprising  how  very  little  work  has  been  done  on  the  protein 
metabolism  of  leaves. 

Deleano's-  investigations  show  that  the  total  nitrogen-content 
of  leaves  of  Vitis  vinifera  did  not  change  appreciably  during  493 
hours  of  respiration  in  the  dark.  He  also  found  but  very  slight 
variation  in  the  protein  nitrogen  up  to  87  hours.  Thereafter  these 
values  decreased.  These  determinations  were  made  by  means  of 
Stutzer's  method,  so  that  they  represent  coagulable  protein,  and  no 
information  can  be  gained  therefrom  regarding  the  amino  acid- 
protein  relations. 

Whether  a  sharp  differentiation  can  be  drawn  between  living  and 
inanimate  protein,  or  better,  perhaps,  between  protoplasmic  and 
non-protoplasmic  protein,  in  the  leaf  appears  exceedingly  difficult. 
It  has  also  been  impossible  to  determine  in  plants  a  factor  corre- 
sponding to  the  "protoplasmic  mass,"  which  has  an  important  role 
in  the  discussions  of  animal  physiologists  in  considering  the  phe- 
nomenon of  respiration.  What,  finally,  the  function  is  of  the  total 
protein  or  of  the  protoplasmic  proteins  in  plants  is  a  question  on 
which  we  have  no  direct  experimental  evidence.  As  has  been  stated, 
one  of  the  chief  difficulties  in  this  problem  has  been  the  inadequate 
information  that  exists  regarding  the  nature  of  the  proteins  of  leaves. 

Palladin^  has  attempted  to  establish  a  relation  between  the  rate 
of  carbon-dioxid  emission  and  of  the  proteins  in  wheat  seedlings. 
Based  upon  the  old  observations  of  Reinke,*  Zacharias,'^  and 
Schwartz,^   Palladin   assumed   that   the  protoplasmic    proteins    are 

*  Mater,  A.     Agriculturchemie,  Gaerungschemie,  138-139  (1902). 

Cf.  also  Irving,  A.,  and  R.  Hankinson.     Biochemical  Jour.,  3,  87,  1908.     Ivanoff,  N.  N., 

Biochem.  Zeitschr.,  120,  1-80  (1921). 
=  Deleano,  N.     Jahrb.  f.  wiss.  BoL,  SI,  587  (1912). 
'  Palladin,  W.     Recherches  sur  la  correlation  entre  la  respiration  des  plantes  et  les  substances 

azotees  actives.     Rev.  Gen.  de  Bot.,  8,  222-284  (1896). 

*  Reinke,  J.     Stiidien  ueber  das  Protoplasma  (1881). 

*  Zacharias,  E.     Bot.  Zeitung  (1881)  169;  (1883)  209. 

*  Schwartz,  F.     Morphologische  und  chemische  Zusammensetzung  dcs  Prutoplasmas.     1887. 


10  STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 

not  digested  by  gastric  juice.  He  thus  finds  that  in  germinating 
seeds  the  rate  of  carbon-dioxid  emission  is  proportional  to  the 
amount  of  protein  indigestible  in  gastric  juice  when  the  supply 
of  carbohydrates  is  ample.  While  these  studies  are  of  prehminary 
nature  and  the  methods^  arbitrary  and  quite  lacking  in  precision, 
they  are  nevertheless  suggestive  of  many  interesting  relations. 

The  fundamentally  important  questions  in  both  respiration  and 
photosynthesis  are  those  relating  to  the  dynamic  aspect  of  these 
phenomena.  Both  of  these  processes  are  primarily  of  interest 
because  they  represent  the  energy  transfer  of  life  phenomena. 
Without  such  energy  transfers  no  manifestiatons  of  life  could  be 
possible.  Just  as  in  physics  and  chemistry,  energetics  is  being 
recognized  as  the  key  to  both  the  structure  and  behavior  of  matter, 
so  in  physiology  further  development  awaits  a  more  comprehensive 
conception  of  energetics.  For  a  long  time  physiological  chemistry 
consisted  in  the  study  of  the  substances  which  enter  into  this  device 
of  energy  transformation  and  the  products  which  leave  it  in  the  form 
of  excreta;  it  is  now  more  and  more  attempting  to  study  the  chemical 
phenomena  which  underlie  the  activity  of  such  energy  changes. 
By  tradition  the  behavior  of  protoplasm  is  associated  with  the  com- 
plex reactions  of  the  proteins.  The  most  complex  group  of  carbon 
compounds,  it  is  also  by  inference  the  medium  for  the  intricate 
reactions  of  life  processes.  Etymologically,  proteins  are  the  sub- 
stance of  "first  importance."  Perhaps  there  is  here  a  fundamental 
difference  between  plants  and  animals,  but,  at  any  rate,  it  is  very 
doubtful  whether  in  plants  a  preponderant  role  can  be  ascribed  to 
the  proteins.  From  the  chemical  viewpoint  the  conception  of 
living  matter  presents  many  insurmountable  difficulties,  while,  on 
the  other  hand,  the  idea  of  a  complex  of  coordinated  chemical  reac- 
tions taking  place  in  a  material  medium  falls  within  the  domain  of 
modern  physical-chemical  reasoning.  If  "living  matter"  is  pos- 
sessed of  properties  and  forms  of  energy  sui  generis,  the  disciplines 
of  physics  and  chemistry  can  be  of  very  Uttle  aid  in  interpreting  the 
behavior  of  Uving  things.  As  long  as  we  are  working  on  a  basis  of 
physics  and  chemistry  it  is  of  little  value  to  introduce  conceptions 
into  physiology  which  have  no  sound  foundation  nor  direct  analogues 
in  these  sciences.  Thus  to  ascribe  the  energy  of  life  processes  to 
"bio tic  energy"  is  certainly  not  clarifying  our  conceptions  of  these 
processes  on  the  basis  of  physical  science.  The  fact  that  physiolo- 
gists are  frequently  having  recourse  to  the  coining  of  new  phrases, 
not  found  in  the  physical  sciences,  to  describe  life  processes  ought 

•  For  the  action  of  pepsin  and  trypsin  on  plant  protoplasm,  cf.  W.  Beidebmann,  Microchemische 
Beobacthungen  an  den  Blattzellen  von  Elodea.  Flora,  11-12,  604  (1918).  Walter,  H. 
Bioehem.  Zeitachr.,  122,  86-99  (1921). 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS.  1 1 

to  serve  as  a  modulator  to  the  mechanists  and  bring  the  realization 
that  pronouncements  on  the  mechanistic  view  of  life  processes  can  be 
made  only  with  reserve  and  as  partial  truths. 

The  term  ''living  matter"  is  in  a  sense  anthropomorphic,  in  that 
we  ascribe  to  the  matter  of  living  things  attributes  and  qualities 
which  we  recognize  in  our  own  behavior.  This  is  in  a  large  measure 
responsible  for  the  confusion  in  our  conception  of  the  essential  nature 
of  the  processes  in  living  things,  we  having  superimposed  on  the 
matter  of  vegetable  organisms  the  behavior  of  our  own  tremendously- 
complex  system,  including  the  maze  of  mental  and  spiritual  expe- 
riences. The  differences  in  the  two  conceptions  at  first  seem  insig- 
nificant or  subtle,  the  one  of  ''living  matter,"  the  other  of  "life  in 
matter";  but  from  the  experimental  physical-chemical  viewpoint 
the  differences  are  fundamental. 

The  gap  between  li\dng  and  non-living  substances  in  a  cell  has 
never  been  bridged  nor  have  the  differences  ever  been  clearly  defined. 
To  the  proteins  has  been  ascribed  the  function  of  life,  partly  because 
of  their  complex  structure  and  reactions  and  also  because  they  have 
been  found  wherever  vital  phenomena  occur.  That  the  proteins 
form  an  essential  part  of  the  medium  in  which  these  complex  chemical 
reactions,  called  "life,"  take  place,  and  that  the  proteins  or  their 
derivatives  contribute  directly  or  through  catalytic  action  to  these 
reactions,  embodies  a  conception  which  is  compatible  with  our  modern 
physical-chemical  thinking.  This  conception  of  a  relatively  inert 
substance  or  medium  in  which  the  interplay  of  chemical  reactions 
takes  place,  and  through  which  these  always  manifest  themselves 
as  life  activities,  is  advanced,  of  course,  not  as  describing  the  actual 
state  of  affairs,  but  as  a  hypothesis  which  lends  itself  to  experimental 
investigation.  Thus  in  the  plant-cell  the  carbohydrates  and  fats 
serve  as  the  fundamental  sources  of  energy  in  respiration.  The 
nitrogen  derivatives,  with  which  the  plant  deals  most  economically 
as  proteinaceous  compounds,  constitute  an  essential  portion  of  the 
medium  in  which  the  multiplicity  of  reactions  occur.  Furthermore, 
these  proteins,  of  themselves  inert,  are  of  fundamental  importance 
because  of  their  ability,  through  their  decomposition  products — 
the  amino-acids — to  influence  the  enzymatic  reactions.  The  dif- 
ferences in  protoplasm  are  thus  to  be  ascribed  to  the  differences  in 
the  media  in  which  the  various  reaction  complexes  occur.  Pro- 
toplasm is  to  be  regarded  as  a  colloidal  mass  of  varying  composition, 
which  serves  as  a  medium  for  the  manifold  chemical  reactions 
involving  the  breakdown  of  many  molecules,  thus  releasing  energy 
which  may  be  used  for  the  synthesis  of  new  compounds,  which  in 
turn  may  be  incorporated  in  the  colloidal  mass  of  the  substratum. 
Nor  can  such  a  hypothesis  explain  or  define  life.     It  simply  attempts 


12  STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 

to  ascribe  more  directly  the  final  forces  which  induce  changes  in 
living  things  to  the  same  causes  which  activate  the  inorganic  world. 
This  is  the  only  hope  for  a  mechanistic  conception  of  life  processes. 

The  question  naturally  arises  whether  in  metabolic  processes 
which  involve  the  liberation  of  energy  a  continual  decomposition  of 
protein  serves  the  plant  (1)  to  contribute  to  the  total  energy  release 
of  decomposition  by  material  other  than  carbohydrates  and  fats, 
or  (2)  to  provide  material  essential  to  the  proper  functioning  of 
enzymes  and  catalysists,  or  (3)  whether  such  a  continued  decompo- 
sition is  an  unavoidable  accompaniment  of  metabolic  energesis. 
Concerning  the  first  possibility,  it  is  evident  that  the  amount  of 
energy  obtained  by  the  decomposition  of  proteins  would  be  very 
small,  for  this  is  usually  a  cleavage  into  amino-acids,  and,  further- 
more, the  leaf  is  capable  of  re-forming  these  amino-acids  into  proteins 
without  the  direct  utilization  of  an  extraneous  source  of  energy,  i.  e., 
through  the  chemo-sjmthetic  energy  derived  from  the  oxidation  or 
the  breakdown  of  sugars. ^  The  total  gain  in  energy  for  the  plant 
from  the  decomposition  of  proteins  would  therefore  be  nil;  and  unless 
the  decomposition  of  proteins  yields  chemical  energy  of  a  certain 
form  or  at  a  particular  rate,  it  is  difficult  to  see  what  advantage 
would  result  or  what  the  mode  would  be  of  deriving  energy  by  a 
continual  decomposition  and  subsequent  resynthesizing  of  protein 
material.  Of  the  possible  different  forms  of  chemical  energy  referred 
to  we  know  at  present  practically  nothing,  so  that  this  is  quite 
beyond  the  ken  of  physical-chemical  reasoning.  That  the  rate 
of  energy  release  from  protein  decomposition  differs  fundamentally 
from  that  of  carbohydrate-oxidation  can  not  be  assumed;  judging, 
however,  from  the  rate  of  accumulation  of  protein-splitting  products, 
it  would  appear  that  the  carbohydrate  breakdown  proceeds  con- 
siderably faster,  mol  for  mol. 

As  to  the  second  possibility  of  the  function  of  protein  decom- 
position— the  providing  of  material  essential  to  the  functioning  of 
enzymes — the  experiments  hereinafter  described  may  contribute  some 
information.  This  aspect  is  of  interest  on  account  of  the  stimulating 
action  which  amino-acids  exert  on  certain  enzymes,  as  well  as  on 
account  of  the  fact  that  amino-acids,  being  amphoteric  electrolytes, 
i.  e.,  capable  of  uniting  with  both  acids  and  bases,  have  the  power  of 
maintaining  the  hydrogen-ion  concentration  within  definite  limits. 

That  amino-acids  accumulate  in  seedlings  left  in  the  dark  has 
been   known    since   the   time   of   Hartig   and   Boussingault.     This 

'  It  is  quite  well  established  that  leaves  are  capable  of  synthesizing  proteins  from  amino-acids 
and  even  nitrates  in  the  dark  in  the  presence  of  an  abundant  carbohydrate  supply.  Protein 
decomposition  in  the  presence  or  absence  of  oxygen  differs  primarily  in  the  proportion  of 
the  various  amino-acids,  principally  asparagine,  tyrosine,  and  leucine.  Suzuki,  Bot.  Centbl., 
75,  289  (1898);  Zalbski.  W.,  Ber.  d.  deutsch.  bot.  Ges.,  15,  536-542  (1897);  Sapoznikow,  W.. 
Bot.  Centbl.,  63  (1893);  Palladin,  W.,  Ber.  d.  deutsch.  bot.  Ges.,  6,  205-212,  296-304  (1888). 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


13 


accumulation  of  amino-acids  has  been  the  subject  of  numerous  inves- 
tigations and  the  basis  of  much  speculation  regarding  the  function 
and  fate  of  proteins  in  plant  respiration.  ^  Suzuki^  is  also  of  the 
opinion  that  during  the  night  the  proteins  of  the  leaf  are  broken 
down  to  amino-acids  and  that  these  migrate  to  other  parts  of  the 
plant.  This  seems  to  be  very  near  to  the  actual  state  of  affairs, 
although  the  extent  to  which  this  takes  place  is  still  an  undecided 
question.  The  solution  thereof  is  made  very  difficult  because  other 
processes,  such  as  photosynthesis  during  the  day  and  respiration 
at  night,  may  often  mask  any  definite  results  observable  in  the 
analytical  data. 

Since  the  development  of  protein  chemistry  the  improved  methods 
of  isolating  and  determining  the  various  amino-acids  have  made  it 
possible  to  establish  what  compounds  are  formed  in  the  decomposition 
of  the  proteins  of  plants  when  these  are  left  in  the  dark.  Most  of 
the  investigations  have  been  carried  out  on  germinating  seeds,  so 
that  many  of  the  results  are  not  altogether  applicable  to  mature 
leaves.  However,  Schulze  for  many  years  has  been  working  on  the 
amino-acid  content  of  plants  and  he  has  obtained  some  very  valuable 
results.  Seedlings  of  Lupinus  grown  for  14  days  in  the  open  were 
reported  by  Schulze  and  Castoro^  to  contain  nitrogen  compounds, 
as  follows : 

Table  3. 


Total  nitrogen. 

Proteins. 

Asparagine. 

Arginine. 

Cotyledons  (dry  material) 

Stems  (dry  material)       

p.ct, 
7.83 
6.77 
5.40 
6.6 

p.ct. 
14.64 

9.56 
11.22 
24.66 

p.ct. 
17.59 
21.12 
10.23 
6.65 

p.  ct. 
0.14 

'!006 

Roots  (dry  material)     

Leaflets  (dry  material) 

Schulze  attributes  the  great  difference  in  the  asparagine  content 
between  the  stems  (21.12  per  cent)  and  the  leaves  (6.65  per  cent) 
to  the  photosynthetic  action  of  the  leaves,  which  in  the  presence  of 
carbohydrates  convert  the  asparagine  into  proteins.  In  harmony 
with  this  it  is  pointed  out  that  the  protein-content  of  the  leaves  is 
2.5  times  as  great  as  in  the  stems. 

1  Pfeffer,  W.     Untersuchungen  ueber  die  Bedeutung  des  Asparagins  beim  Keimen  der  Samen. 
Jahrb.  f.  wiss.  BoL,  8,  429-574  (1880). 
Meuniek,  F.     fitude  sur  I'asparagine.     Ann.  Agron.,  6,  275-281  (1880). 

Borodin,  J.     Ueber  die  physiologische  Rolle  und  die  Verbreitung  des  Asparagins  im  Pflanzen- 
reiche.     Bot.  Zeit.,  36,  801-832  (1878). 
»  Suzuki,  W.     Bull.  Coll.  Agr.  Tokyo  2,  458  (1896). 

'  Schulze,  E.,  and  N.  Castoro.     Beitrege  zur  Kenntniss  der  Zusammensetzung  und  des  Stoff- 
wechsels  der  Keimpflanzen.     Zeit.  physiol.  chem.,  38,  244  (1903). 
Also  KiESEL,  A.,  Zeit.  physiol  chem.,  49, 72-80  (1906) ;  Andr6,  G.,  Compt.  rend.,  148, 1685-1687; 
Thompson,  S.  G.,  Jour.  Amer.  Chem.  Soc.,  37  230-235  (1915). 


14 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


In  our  own  experiments  with  mature  leaves  of  Helianthus  annuus 
we  found  that  the  amino-acid  content  of  the  leaves  was  higher  than 
that  of  the  stems,  while  the  carbohydrate-content  was  considerably 
higher  in  the  stems. 

Comparing  seedUngs  which  had  developed  in  the  light  with  those 
grown  in  the  dark,  Schulze  and  Castoro  found  that  the  amount  of 
amino-acids  in  etiolated  seedlings  7  days  old  was  higher  than  in  the 
green  plants  twice  as  old,  while  the  amount  of  asparagine  was  about 
the  same  in  both.  In  general,  it  would  seem  from  these  investigations 
that  the  amount  of  amino-acid  formation  and  protein  breakdown  is 
considerably  reduced  when  there  are  carbohydrates  present  or  the 
power  of  carbohydrate  formation  through  photosynthesis  is  available 
to  the  seedlings.  This  substantiates  the  general  principle  that 
carbohydrates  are  protein-sparers,  which  has  been  found  by  other 

Table  4. — Distribution  of  nitrogen  compounds  in 
leaves  and  stems  according  to  Schidze's  analysis. 


In  leaves. 

In  stems. 

62.56 

8.06 

21.46 

7.92 

23.04 

6.20 
66.17 
4.59 

Phosphotungstic-acid 

precipitate 

Asparagine 

Other  compounds 

investigators.  1  These  studies  by  Schulze  have,  however,  only  an 
indirect  bearing  on  our  investigations,  because  Schulze  was  dealing 
largely  with  the  germination  of  seeds  in  which  reserve  proteinaceous 
material  was  in  the  course  of  the  metabolic  activity  of  the  plant 
resolved  into  simpler  compounds,  and  the  plants  were  not  mature 
self-supporting  organisms.  Schulze^  has  made  some  extensive 
investigations  of  the  amino-acid  transformations  in  young  plants. 
In  seedlings  of  the  Vicia  saliva,  Pisum  sativum,  Lwpinus  luteus, 
and  Lupinus  alhus  grown  in  the  dark  there  occurs  a  marked  accumu- 
lation of  amino-acids.  The  nature  of  the  amino-acids  changes  with 
the  length  of  time  the  plants  are  allowed  to  remain  in  the  dark. 
Seedlings  left  in  the  dark  6  to  7  days  contain  leucin,  asparagine, 
arginine,  histidine,  lysine,  and  tyrosine  in  small  quantities.  After 
remaining  in  the  dark  for  longer  periods,  2  to  3  weeks,  leucine,  tyro- 
sine, and  arginine  decrease,  while  the  amount  of  asparagine  is  greatly 
increased.  Schulze  considers  leucine,  arginine,  histidine,  and  lysine 
as  the  primary  products  of  the  protein  breakdown  in  leaves,  and  that 
these  substances  in  turn  are  converted  into  asparagine  and  glutamine 


»  Deleano,  N.  T.     Jahr.  f.  wiss.  BoL,  SI,  541-592  (1912). 

*  Schulze,  E.     Ueber  den  Umsatz  der  Eiweissstoffe  in  der  lebenden  Pflanze. 
chem.,  30,  241-312  (1900). 


Zeit.   physio. 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS.  15 

after  longer  periods  of  darkness.  He  considers  that  asparagine  is 
used  in  the  leaves  for  protein  synthesis.  This  is  concluded  from  the 
smaller  quantities  of  asparagine  found  in  the  leaves. 

In  our  own  experiments  with  germinating  beans  (see  page  40), 
some  in  the  dark  and  some  in  the  light,  we  found  but  slight  difference 
between  the  "light"  and  ''dark"  plants.  However,  these  experi- 
ments were  run  only  120  hours,  while  in  the  investigations  of  others, 
already  referred  to,  the  analyses  were  made  after  periods  of  1  to  4 
weeks. 

The  experiments  with  mature  leaves  which  are  described  hereafter 
show  clearly  that  in  the  dark  there  is  an  accumulation  of  amino 
acids.  This  is  true  not  only  of  the  excised  leaves  but  also  of  those 
attached  to  the  plant.  How  intimate  a  causal  interrelationship 
exists  between  amino-acid  accumulation  and  carbohydrate  depletion 
it  is  impossible  to  state  from  the  experimental  data  as  yet  available. 
This  is,  of  course,  an  extremely  complicated  problem,  and  requires 
for  solution  much  deeper  knowledge  of  the  nature  and  function  of 
plant  proteins  than  we  are  now  in  possession  of.  This  point,  how- 
ever, does  become  evident,  that  the  amino-acids  have  a  profound 
influence  on  the  rate  of  respiratory  activity  of  the  leaves,  in  the 
sense  that  an  increase  in  amino-acid  content  is  accompanied  by  an 
increase  in  carbon-dioxid  emission  and  carbohydrate  consumption. 

In  compendium,  then,  it  appears  that  in  mature  leaves: 

1.  Amino-acids  accumulate  in  the  dark  as  carbohydrates  are  consumed.  There 
is  some  evidence  that  proteins  also  diminish  in  this  process. 

2.  The  rate  of  carbohydrate  consumption  is  accelerated  by  amino-acids.  At  the 
same  time  the  fact  must  not  be  neglected  that  respiratory  activity  is  a  product 
(within  limits)  of  mass  action,  the  rate  depending  as  one  factor  upon  the  supply  of 
available  carbohydrates. 

The  questions  then  presented  are:  In  what  manner  can  amino 
acids  influence  the  rate  of  respiratory  activity?  What  is  the  influence 
of  these  substances  on  the  general  carbohydrate  metabolism?  Are 
there  any  chemical  relations  between  amino-acids  and  sugar  which 
can  account  for  this  behavior,  or  is  this  influence  exercised  through 
the  operation  of  enzymes?  Contributing  to  the  elucidation  of  these 
questions  are  a  number  of  facts  which  can  be  drawn  from  allied 
sciences. 

Of  direct  bearing  on  the  question  of  the  influence  of  amino-acids 
on  the  respiratory  activity  is  the  phenomenon  of  the  specific  dynamic 
effect  of  proteins,  which,  observed  in  animals,  has  been  the  subject 
of  extensive  experimental  investigation  and  much  controversy. 
Rubner  and  others  established  that,  in  the  diet  of  animals,  fats  and 
carbohydrates  are  mutually  interchangeable  on  a  calorific  basis. 
Thus  100  grams  of  fat  can  be  replaced  by  232  grams  of  starch  and 
by  234  grams  of  saccharose.     In  attempting  to  apply  this  principle  of 


16 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


isodynamic  values  to  proteins,  a  number  of  difficulties  were  encoun- 
tered. It  was  found  that  an  increase  of  protein  in  the  diet  stimulates 
the  total  metaboUsm,  so  that  actually  more  food  is  utilized  and  more 
heat  emitted  from  a  diet  rich  in  protein  than  from  one  containing 
little  protein.  Rubner^  subjected  this  phenomenon  to  a  very 
thorough  and  painstaking  investigation  and  gave  it  the  name  of 
specific  dynamic  action  of  proteins.  He  found  that  the  increase  in 
metabolic  activity  was  greater  with  proteins  than  with  any  other 
class  of  foods  and  that  dogs  fed  on  meat  respired  more  actively 
without  doing  any  external  work.  After  establishing  the  relation 
of  environmental  temperature  to  metaboUc  rate  and  excluding  the 
reflex  increase  in  metabolism  through  chemical  regulation,  Rubner 
was  able  to  determine  the  true  values  of  the  specific  dynamic  action 
of  proteins.  The  ingestion  of  the  starvation  requirement  for  the 
various  forms  of  food  raised  the  metabolism  as  shown  in  table  S.^ 


Table  5. 

Diet. 

Heat 
increase. 

Meat 

p.ct. 

36.7 

12.7 
5.0 

13.4 
4.0 
4.0 

21.5 

Fat 

10  per  cent  meat,  90  per  cent  fat. . . 
Meat,  fat,  sugar 

Meat,  fat,  starch     

20  per  cent  meat,  80  per  cent  fat. . . 

A  great  advance  in  the  understanding  of  the  specific  dynamic 
action  of  proteins  was  made  by  the  investigations  of  Lusk.^  Instead 
of  using  proteins  Lusk  fed  amino-acids.  The  same  action  is  observ- 
able with  these  splitting  products  of  the  proteins.  It  was  found 
that  glycocoU  and  alanine  greatly  increase  the  metabolic  activity,  that 
leucin  and  tyrosine  exert  but  a  slight  effect,  and  that  glutamic  acid 
is  without  effect.  These  findings  are  especially  interesting  in  view 
of  the  following  facts:  It  is  known  that  glycocoU  and  alanine  are 
completely  convertible  into  glucose  in  the  diabetic  organism,  whereas 
glutamic  acid  is  so  converted  that  three  of  its  carbons  go  to  form 
glucose,  while  the  other  two  carbon  atoms  are  oxidized.  When 
glycosuria  is  artificially  produced  by  giving  phlorihizin,  and  the 

'  Rubner,  Max.     Die  Gesetze  des  Energieverbraucha  bei  der  Ernaehrung  (1902). 

*  Rubner,  Max.     I.  c,  325. 

'  Lusk,  Graham,  and  S.  A.  Riche.     Animal  calorimetry.     V,  Influence  of  the  ingestion  of 
amino-acids  upon  metabolism.     Jour.  Biol.  Chem.,  13,  155-183  (1912). 
Lusk,  Graham.     The  cause  of  the  specific  dynamic  action  of  protein.     Arch.  Intern.  Med., 
12,  485-487  (1914).     Animal  calorimetry:    XI.  An  investigation  into  the  causes  of  the 
specific  dynamic  action  of  the  foodstuffs.     Jour.  Biol.  Chem.,  20,  555-617  (1915). 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


17 


dog  is  given  glycocoll,  there  is  no  oxidation  of  this  material,  the  energy- 
content  of  the  glycocoll  is  eliminated  in  the  urine  as  glucose  and  urea, 
nevertheless  the  metabolism  is  greatly  increased.  When  corre- 
sponding or  even  larger  quantities  of  glucose  are  given  there  is  no 
stimulation  of  metabolism.  From  this  it  follows  that  the  immediate 
cause  of  the  specific  dynamic  action  is  not  in  the  oxidation  of  the 
amino-acids  and  liberation  of  energy  therefrom,  but  that  the  amino- 
acids  in  some  manner  stimulate  the  activity  of  the  cells  and  thus 
cause  them  to  metabolize  more  food  material. 

The  specific  dynamic  action  of  proteins,  especially  with  the  insight 
gained  through  Lusk's  investigations,  throws  considerable  light  on 
the  general  problem  of  the  function  of  amino-acids  in  energesis. 
It  does  not,  however,  enlighten  us  on  the  fundamental  causes  of  this 
stimulating  action  of  the  amino-acids. 

Our  first  inclination  was  to  search  for  a  purely  chemical  explanation 
which  could  account  for  this  remarkable  behavior.  A  natural 
supposition  seemed  to  be  that  the  amino-acids  affected  the  sugars  in 
such  a  manner  as  to  make  them  more  easily  broken  down  and  oxi- 
dized. As  Nef^  has  shown,  glucose  is  a  relatively  stable  sugar, 
while  fructose  is  more  completely  oxidized.  Thus,  in  order  to  reduce 
completely  a  mixture  of  382.5  grams  CUSO4  and  163.8  grams  NaOH 
(Fehling's  solution),  the  amounts  of  the  various  sugars  shown  in 
table  6  were  required: 

Table  6. 


Sugar. 

Grams  required 
for  reduction. 

Corresponding  to 
atoms  of  oxygen. 

Arabinose .... 

Mannose 

Dextrose 

Levulose 

55.0 
66.4 
68.0 
64.0 

2.13 
2.45 
2.39 
2.16 

Similarly,  Lusk^  demonstrated  that  fructose  was  metabolized 
more  actively  than  glucose,  so  that  the  percentage  of  increase  over 
the  indirect  basal  metabolism  for  the  various  sugars  was  found  to  be : 
glucose  30,  fructose  37,  sucros6  34,  galactose  22,  and  lactose  3. 
In  view  of  these  facts  it  was  possible  that  the  amino-acids  acted  as 
isomerizing  agents,  converting  the  glucose  into  the  more  active 
fructose,  just  as  had  been  shown  to  happen  with  a  great  variety 
of  substances,  e.  g.,  Ca(0H)2,  Pb(0H)2,  NaaCOs,  etc.^  All  of  our 
experiments  directed  to  estabHsh  such  an  isomerizing  action  of  the 
amino-acids   on   various   hexose   sugars  yielded    negative    results. 

1  Nep,  J.  U.     Annalen  der  Chemie     (Leibig),  357,  219  (1908). 

«  LuBK,  Graham.     Jour.  Biol.  Chem.,  20,  656  (1915). 

»  Nef,  J.  U.     Annalen  der  Chemie  (Liebig),  357,  294-312  (1908);  403,  240-383  (1914). 


18  STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 

Furthermore,  while  fructose  fed  to  mature  leaves  showed  relatively 
high  respiratory  activity,  we  were  surprised  to  find  that  the  respir- 
atory activity  of  the  leaves  fed  fructose  was  apparently  depressed 
when  amino-acids  were  also  given. 

A  more  encouraging  possibility  for  an  explanation  of  the  stimula- 
tory action  of  amino-acids  was  offered  by  the  consideration  of  the 
influence  of  these  substances  on  enzyme  activity.  That  some  amino 
acids  have  a  decided  effect  on  the  rate  of  activity  of  certain  enzymes 
has  been  known  for  a  long  time.  Effront^  in  1893  reported  that 
asparagine  accelerates  the  hydrolysis  of  starch  by  takadiastase  and 
later^  showed  that  this  accelerating  action  was  also  accomplished 
by  other  amino-acids,  while  amines  and  their  salts  and  acid  amides 
and  their  salts  showed  no  such  action. 

The  observation  that  the  saccharification  of  starch  by  pancreatic 
juice  was  accelerated  by  amino-acids  was  reported  by  Terroine 
and  Weill. ^  Even  very  low  concentration  of  gylcocoll,  1:10,000, 
increases  the  velocity  of  the  saccharification  30  to  40  times.  Dakin^ 
observed  that  many  condensations  which  have  their  analogues  in 
the  metabolism  of  living  organisms  are  induced  by  amino-acids, 
peptones,  albuminose,  and  some  proteins,  while  the  same  reactions 
do  not  take  place  without  these  substances.  Jacoby  and  Umeda^ 
also  observed  that  the  activity  of  soja-urease  is  greatly  accelerated 
by  glycocoll,  anahne,  tyrosine,  leucin,  and  glutamic  acid.  The 
activity  of  pine  diastase  was  investigated  by  Ujhara,"  who  found 
that  this  action  was  increased  48.5  per  cent  by  very  small  quantities 
of  glutamic  acid  and  by  glycocoll  45.1  per  cent.  Similar  results  were 
obtained  with  dog-serum  diastase.  Sherman  and  Walker^  have 
subjected  the  accelerating  action  of  amino-acids  on  diastase  to  thor- 
ough investigation.  They  have  found  the  acceleration  to  take  place 
with  diastase  derived  from  a  variety  of  sources  as  well  as  with  starches 
of  widely  different  origin.  Also,  the  acceleration  is  not  due  to  any 
change  in  the  hydrogen-ion  concentration  nor  to  a  more  favorable 
concentration  of  the  electrolytes. 

Finally,  Burge^  has  investigated  the  influence  of  introducing 
various  amino-acids  into  the  intestine  and  stomach  on  the  catalase 
activity  of  the  blood.  While  this  is  stimulated  apparently  by  a 
large  variety  of   substances,  it  would  seem  that  the  amino-acids 

»  Effront,  I.     Mon.  Sci.,  41,  266  (1893). 

*  Idem.     Ibid.,  (4),  18,  561  (1904). 

»  Terroine,  E.,  and  J.  Weill.     Chem.  Abstracts,  4,  1535  (1913). 

*  Dakin,  H.  D.     Jour.  Biol.  Chem.,  7,  49-55  (1910). 

*  Jacoby,  M.,  and  N.  Umeda.     Biochem.  Zeitschr.,  68,  23-47  (1918). 
«  Ujhara,  K.     Chem.  Abstracts,  12,  1971  (1918). 

'  Sherman,  H.  C,  and  Florence  Walker.  Influence  of  aspartic  acid  and  asparagine  upon  the 
enzymatic  hydrolysis  of  starch.  Jour.  Amer.  Chem.  Soc,  41,  1866-1873  (1919);  43,  2461- 
2476  (1921). 

«Burqe,  W.  E.  Amer.  Jour.  Physiol.,  47,  351-355  (1918);  48,  133-140  (1919);  Science,  n.  s., 
49,  594-595  (1919). 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS.  19 

are  more  effective  in  this  respect  than  any  of  the  other  substances 
tried,  i.  e.,  sodium  acetate,  acetamide,  glycerine,  oleine,  and  dextrose. 

It  must  be  reaUzed,  of  course,  that  the  evidence  regarding  the 
behavior  of  amino-acids  toward  enzymes,  here  introduced,  does  not 
serve  as  a  direct  explanation  of  the  influence  of  amino-acids  on  the 
rate  of  respiratory  activity  nor  of  the  specific  dynamic  action  of 
proteins.  However,  in  the  present  very  incomplete  state  of  our 
knowledge  of  this  subject,  the  information  of  these  relations  may 
serve  as  a  valuable  guide  in  developing  further  our  conceptions  of 
the  respiratory  processes.  So,  while  there  is  as  yet  no  simple  expla- 
nation of  the  role  of  amino-acids  in  respiration,  it  seems  also  at 
present  exceedingly  doubtful  whether  such  a  process  is  amenable 
to  simple  treatment. 

We  are  obliged  to  recognize  more  and  more  that  not  only  are  the 
various  activities  of  a  living  organism  intimately  interrelated, 
but  that  the  proper  functioning  of  any  one  process  depends  upon 
the  coordination  of  various  enzymatic  activities.  Thus  in  the  oxy- 
biosis  of  carbohydrates  there  are  naturally  a  great  many  steps  be- 
tween the  stored  starch  and  the  formation  of  carbon  dioxid  and 
water.  Plant  physiologists  have  very  generally  fallen  into  the 
habit  of  overemphasizing  and  reasoning  as  to  the  modus  vivendi 
of  reactions  in  living  things  from  evidence  of  the  substances  found  in 
such  organisms.  It  must  be  borne  in  mind  that,  applying  the  prin- 
ciples of  the  kinetics  of  step  reactions,  the  rate  of  the  whole  reaction 
is  determined  by  the  rate  of  the  step  progressing  with  the  lowest 
speed,  and  the  more  rapidly  a  total  reaction  progresses  the  fewer 
intermediate  products  are  there  possible  to  detect.  Thus,  often 
the  most  important  and  reactive  products  are  not  detectable  by  our 
present  chemical  methods. 

Fundamentally  a  better  understanding  of  respiration  depends 
upon  a  knowledge  of  the  nature  of  enzyme  activity,  and  this,  of 
course,  brings  us  to  the  very  frontier  of  scientific  thought.  While 
the  activity  or  mode  of  action  of  enzymes  has  its  analogy  in  more  or 
less  well-defined  behavior  of  catalysis,  the  composition  of  the  enzymes 
themselves  is  an  open  question.  Briefly,  there  appear  to  be  two 
schools — the  one  conceiving  of  an  enzyme  as  a  definite  complex 
substance  with  catalytic  properties,  the  other  regarding  enzymes 
not  as  chemical  individuals  but  rather  as  mixtures  or  complex 
systems.  Although  the  former  conception  has  received  very  little 
advancement  through  the  efforts  of  synthetic  chemistry,  it  has  its 
strong  adherents,  as,  for  instance,  Willstaetter,^  who  hopes  by  means 
of  preparation  work  to  obtain  constantly  purer  products  until  their 
chemical  nature  can  be  established.  On  the  other  hand,  following 
the  idea  of  enzymes  as  complex  mixtures,   some  very  suggestive 

»  WiLLSTAETTEB,  R.     Zeitsch.  f.  atigew.  chem.,  33,  209  (1920). 


20  STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 

results  have  recently  been  obtained  which  are  pertinent  to  the 
general  subject  of  the  role  of  amino-acids  in  respiration. 

Various  claims  have  been  advanced  from  time  to  time  regarding 
the  artificial  production  of  enzymes,  and  a  number  of  catalytic 
reactions  have  been  produced  by  means  of  these  products — reactions 
which  are  very  closely  analogous  to  those  produced  by  enzymes. 
In  the  extensive  literature  on  this  subject  a  very  serious  error  has 
been  allowed  to  enter — a  matter  of  inference  originally  rather  than 
of  direct  statement.  It  is  a  very  debatable  question  whether  the 
results  with  these  "artificial  enzymes,"  i.  e.,  mixtures  of  substances 
which  induce  certain  reactions,  which  are  also  produced  by  true 
enzymes,  are  of  such  great  value  in  revealing  the  composition  of  the 
enzymes  as  has  been  claimed.  It  appears  rather  that  these  reactions 
can  be  induced  by  a  variety  of  catalyses,  as  has  been  found  to  be 
the  case  for  many  inorganic  and  organic  reactions,  and  simply  that 
it  has  been  possible  for  chemists  to  produce  one  of  these  catalysts. 
However,  it  does  not  now  follow  that  this  is  necessarily  the  same 
catalyst  which  is  produced  and  used  by  the  organism  in  the  form  of 
an  enzyme.  The  point  is  essentially  one  of  interpretation  of  experi- 
mental results. 

Nevertheless,  the  results  obtained  with  these  various  "artificial 
enzymes"  are  of  great  value  in  explaining  catalytic  action,  and 
therefore  are  of  direct  bearing  on  enzyme  activity.  Herzfeld*  has 
shown  that  the  action  of  the  proteolytic  enzymes,  pepsin  and 
trypsin,  can  be  simulated  by  a  prepared  mixture  of  amino-acids  and 
polypeptides.  According  to  this  author's  views,  autolysis  is  simply 
an  autocatalysis  which  is  started  by  the  introduction  into  the  system 
of  some  of  the  products  of  decomposition.  Baur  and  Herzfeld^ 
have  carried  these  researches  further,  and  announce  a  splitting  of 
glucose  into  alcohol  and  carbon  dioxid  by  means  of  carefully  prepared 
mixtures  containing  peptone,  bile  salts,  dextrin,  and  sodium  bicar- 
bonate. Although  the  proportion  of  carbon  dioxid  and  alcohol 
formed  in  these  reactions  does  not  correspond  to  that  obtained  in 
zymase  fermentation,  the  authors  explain  this  by  the  fact  that  their 
catalyst  also  produces  other  sugar  decompositions.  Provided  the 
toluene  employed  to  prevent  infection  by  living  organism  was 
thoroughly  efficacious,  which  is  open  to  some  doubt,  these  experiments 
point  the  way  to  a  very  fruitful  field  in  the  investigation  of  enzyme 
activity. 

>Herzfeld,  E.,  Biochem.  ZeUschr.,  64,  103-105  (1914);  68,  402-435  (1915);  88,  260  (1918). 
*  Baur,  E..  and  E.  Herzfeld.     Biochem.  Zeitschr.,  117,  96-112  (1921). 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS.  21 

METHODS  AND  APPARATUS. 

1.  The  Experimeiital  Material. 

For  the  study  of  respiration  and  photosynthesis  the  leaves  of  all 
plants  are  not  equally  well  suited.  That  is,  in  some  plants,  due  to 
structural  peculiarities,  these  processes  exhibit  complications  and 
complexities  which  make  the  interpretation  of  observational  data 
extremely  difficult.  The  fundamental  feature  of  both  processes 
is  that  they  depend  upon  the  ingress  and  egress  of  gases.  The  most 
satisfactory  methods  of  measuring  the  rate  of  respiration  and  photo- 
synthesis are  based  upon  the  gaseous  exchange,  and  any  factor 
which  at  all  influences  the  easy  diffusion  of  these  gases  interferes 
with  the  accuracy  of  the  determinations.  For  this  reason,  thin 
leaves  are  more  easily  worked  with  than  those  which  are  succulent 
or  possess  protective  devices  against  the  loss  of  water. 

Moreover,  in  a  study  of  respiration,  analytical  data  are  of  great 
importance  and  the  leaf  material  should  not  present  undue  diffi- 
culties for  the  chemical  determination  of  the  materials  intimately 
associated  with  the  respiratory  activity.  The  plant  material  should 
also  be  capable  of  being  easily  grown  in  quantity  and  should  not  be 
susceptible  to  infection  or  disease.  Further  insight  into  the  mechan- 
ism of  the  processes  of  respiration  and  photosynthesis  can  be  gained 
only  by  means  of  experimentation,  that  is,  by  subjecting  the  plant 
to  a  variety  of  external  conditions  and  at  will  altering  some  of  the 
internal  constituents.  For  such  a  procedure  the  plant  must  not  be 
too  sensitive  nor  easily  affected  by  shght  variations  of  external 
conditions.  In  view  of  the  fact  that  these  studies  were  directed 
primarily  toward  an  attack  on  the  problem  of  photosynthesis,  it 
was  desirable  to  work  with  leaves  in  which  the  chlorophyll  apparatus 
was  well  developed,  and  that  these  leaves  should  be  autotrophic  in 
the  sense  that,  aside  from  mineral  nutrients,  they  should  be  entirely 
capable  of  producing  their  own  food  material. 

It  would,  of  course,  be  desirable  to  gain  information  relative  to 
the  respiratory  and  photosynthetic  processes  of  a  large  variety  of 
species.  We  are  fully  aware  that  different  species  exhibit  great 
variation  in  their  behavior.  Similarly,  a  knowledge  of  the  behavior 
of  these  plants  in  the  field,  under  natural  conditions,  would  probably 
be  of  greater  significance  than  the  information  gained  under  such 
highly  artificial  conditions.  However,  these  ideals  are  not  amenable 
to  the  experimental  method  and  can  be  attempted  only  at  too  great 
a  sacrifice  of  precision  and  intensity  of  investigation. 

We  were  therefore  constrained  to  confine  our  studies  to  a  very 
limited  number  of  plants  which  fulfilled  the  requirements  mentioned. 
The  wild  sunflower  of  southern  Arizona,  Helianthus  annuus,  and 


22  STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 

the  "Canada  Wonder"  bean  have  answered  the  requirements 
admirably,  and  we  have  been  able  to  grow  this  material  in  the  green- 
house during  most  of  the  year.  Naturally,  care  was  exercised  to 
use  only  perfect  leaves,  free  from  infections  and  other  imperfections. 
By  thus  concentrating  our  efforts  we  hoped  to  be  able  to  gain  deeper 
and  more  detailed  information  which  might  serve  to  elaborate  some 
general  principles  of  plant  metabolism. 

For  most  of  the  experiments  leaves  which  had  been  cut  from  the 
plant  were  used.     The  method  of  cutting  is  described  later. 

Although  experiments  with  excised  leaves  apparently  represent 
highly  artificial  conditions,  this  method  possesses  many  distinct 
advantages.  An  entire  plant  usually  requires  so  much  space  that 
it  is  exceedingly  difficult  and  cumbersome  to  construct  respiration 
chambers  which  will  accommodate  the  plant  and  at  the  same  time 
permit  accurate  control  of  the  temperature.  By  using  excised 
leaves  the  space  can  be  very  materially  reduced  and  the  requirements 
of  temperature  control  adequately  met.  A  relatively  small  volume 
of  the  respiration  chamber  has  another  advantage  in  that  with  a  given 
rate  of  the  air-stream  it  permits  the  more  perfect  removal  of  the 
gases.  By  cutting  the  leaf  from  the  plant  the  factor  of  translocation 
is  also  eliminated.  Thus  materials  which  are  formed  in  the  leaf 
during  the  course  of  respiration  do  not  migrate  to  other  parts  of  the 
plant,  and  the  only  source  of  supply  of  food  material  is  that  which  is 
stored  in  the  leaf  itself.  The  general  effect  of  these  conditions  as 
compared  with  the  attached  leaves  is,  as  it  were,  to  shorten  the  time 
of  certain  reactions  or  accentuate  their  intensity.  By  removal  from 
the  rest  of  the  plant,  the  leaf  has  thus  been  severed  from  its  base 
of  supplies  as  well  as  from  the  receiver  of  its  surplus  products. 

The  use  of  excised  leaves  also  offers  the  only  satisfactory  method 
of  feeding  to  leaves  substances  the  behavior  of  which  it  is  desired  to 
study.  In  comparison  with  animals,  physiological  work  with  plants 
is  at  a  greater  disadvantage  in  this  respect.  It  is  not  possible  to 
ingest  into  plants  a  definite  quantity  or  kind  of  food  material,  as 
can  be  done  with  animals.  However,  it  has  been  established  that 
many  organic  substances  are  taken  up  into  the  leaves  in  a  short  time 
when  the  petioles  are  placed  in  solutions  of  these  substances.  On 
the  other  hand,  under  the  conditions  of  our  experiments  there  was 
no  migration  of  materials,  such  as  sugars,  from  the  leaf  into  the 
nutrient  solution. 

The  plants  used  in  these  investigations  were  grown  in  a  green- 
house in  a  loam  soil  to  which  no  fertilizer  had  been  added.  Care 
was  taken  to  use  leaves  of  about  the  same  age,  size,  and  development. 
It  was  endeavored  to  study  the  behavior  of  mature,  well-functioning 
leaves;  so  both  rapidly  growing  and  old  leaves  were  discarded. 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


23 


2.   The  Apparatus. 

The  determinations  of  the  rate  of  respiration  were  made  on  the 
principle  of  drawing  air  free  from  carbon  dioxid  over  the  leaves 
and  absorbing  the  carbon  dioxid  given  off  by  the  leaves  in  a  stand- 
ardized solution  of  barium 
hydroxide.  The  appara- 
tus  was  so  arranged  that 
the  leaves  were  always 
under  the  pressure  of  the 
atmosphere.  A  small  pis- 
ton-pump driven  by  an 
electric  motor  drew  the 
air  through  the  entire  ap- 
paratus; a  large  reservoir 
and  a  pressure  regulator 
assured  the  regularity  of 
the  stream.  The  air  was 
drawn  from  out  of  doors 
in  order  to  avoid  the  pos- 
sibility of  deleterious  ef- 
fects from  impurities  in 
the  air  of  the  laboratory. 
It  was  passed  first  through 
a  train  of  soda-lime  tubes 
to  remove  the  carbon  di- 
oxid and  then  through  a 
coil  of  metal  tube,  10  feet 
long,  which  was  immersed 
in  the  water  thermostat. 
A  large  Freas  electric  ther- 
mostat was  employed  and 
set  at  exactly  25°.  From 
the  metal  tube  the  air 
passed  to  the  upper  open- 
ing of  the  respiration 
chamber. 

A  drawing  of  the  respi- 
ration chamber  is  given 
in  figure  1.  This  con- 
sists essentially  of  a  gal- 

vanized-iron  can  40  cm.  deep  and  18  cm.  in  diameter.  A  heavy 
lead  base  permits  the  entire  chamber  to  be  submerged  in  the 
water  of  the  thermostat.  To  the  top  of  the  chamber  is  sol- 
dered a  collar  which  forms  a  trough.     Into  this  trough  fits  loosely 


Figure  1. 
Respiration  chamber  with  mercury  seal  and  cover,  with 
broken  out  section  showing  container  for  nutrient  solu- 
tion and  device  for  holding  petioles  of  leaves. 


CSi«^ 


24 


If- 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


an  inverted  glass  crystallizing  dish  and  over  this  a  galvanized- 
iron  cover.  As  a  seal,  the  trough  contains  mercury  about  3  cm. 
deep  and  over  this  is  placed  water.  In  order  to  protect  the 
trough  and  metal  cover  against  the  action  of  the  mercury,  these 
were  first  electroplated  with  copper,  then  covered  with  nickel,  and 
finally  given  a  coat  of  waterproof  lacquer.  When  the  respiration 
chamber  is  in  use  it  is  entirely  covered  by  the  water  of  the  thermostat 
and  quickly  attains  the  temperature  of  the  bath.     The  metal  cover 


Figure  2. 
Apparatus  for  automatically  changing  the  course  of  the  air-stream  from  one  absorbing  tube 
to  another.  The  bell-valve  with  mercury  seal  is  opened  by  means  of  the  electromagnetic  coil 
and  closed  when  the  current  is  cut  out  of  the  coil.  One  valve  is  attached  to  each  coil.  In  the 
cut,  one  complete  valve  connected  with  the  electromagnet  which  controls  it  is  shown;  the  three 
other  coils  are  indicated. 

can  be  removed  during  the  course  of  an  experiment  to  observe  the 
leaves  in  the  chamber  or  to  admit  light,  as  the  glass  cover  in  the 
mercury  makes  an  absolutely  tight  seal. 

On  the  bottom  of  the  chamber  is  a  glass  dish  containing  a  large 
number  of  short  pieces  of  glass  tubing  set  on  end.  This  dish  contains 
the  nutrient  solution  and  the  glass  tubes  are  of  a  size  to  admit  easily 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


25 


the  petioles  of  the  leaves;  one  leaf  in  position  is  shown  in  the  drawing. 
Fresh  sterilized  solutions  are  of  course  used  for  each  experiment, 
the  entire  chamber  and  dish  being  sterilized  each  time. 

The  air-stream  enters  the  chamber  through  the  upper  tube  and 
leaves  through  the  lower  one.  To  the  latter  a  trap  is  attached  to 
catch  the  water-vapor  which  is  carried  out  and  condenses  in  the 
tubes.  By  means  of  glass  tubing  the  air-stream  is  distributed 
through  four  glass  stopcocks  to  the  four 
mercury  bell-valves.  These  valves  are 
electrically  controlled  by  means  of  a 
clock  and  require  detailed  description. 

Our  preliminary  studies  on  the  res- 
piration of  leaves  soon  revealed  the 
necessity  of  making  carbon-dioxid  deter- 
minations over  a  longer  period  of  time 
than  had  usually  been  done.  From  ob- 
servations of  a  few  hours  or  a  whole  day 
only  limited  information  can  be  gained 
as  to  the  true  nature  of  this  process, 
especially  if  the  leaves  are  to  be  fed 
various  substances.  Moreover,  ^5  too 
long  periods,  as  during  an  entire  night, 
are  for  obvious  reasons  to  be  avoided. 
These  requirements  necessitated  atten- 
tion being  given  to  the  experiments 
during  the  entire  24  hours  of  the  day. 
Therefore,  in  order  to  obviate  the  night 
work,  an  apparatus  was  devised  which 
automatically  changed  the  course  of  the 
air-stream  from  one  absorbing  tube  to 
another  at  definite  times. 

As  was  stated,  the  air-stream  passes 
from  the  distributing  tubes  to  the  mer- 
cury valves.  The  construction  of  these 
valves,  together  with  the  electric  coils 
which  control  them,  is  pictured  in  figure 
2;  a  detail  of  the  mercury  valve  is  shown  in  figure  3.  In  figure  2 
the  valve  is  show^n  open,  giving  free  passage  of  the  air-stream  from 
the  distributing  tube  through  the  valve  to  the  absorbing  tubes.  The 
valve  is  connected  by  means  of  a  heavy  wire  to  a  piece  of  laminated 
iron  which  forms  the  core  of  an  electric  magnet.  When  the  current 
passes  through  the  wire  coil,  the  core  is  drawn  up  and  the  valve 
remains  open  as  long  as  the  current  passes  through  this  coil.  As  soon 
as  the  current  stops  passing  through  the  coil,  the  iron  core  drops 
and  the  valve  is  closed  by  means  of  the  glass  tube  which  is  immersed 


Figure  3. 
Detail  of  bell- 
valve,  made  of  glass 
and  sealed  with  mer- 
cury. The  upper 
portion  of  the  glass 
tube  is  attached  to 
the  core  of  the  elec- 
tromagnet; the  lower 
end  is  immersed  in 
mercury  when  the 
valve  is  closed  and 
drawn  out  of  the 
mercury  by  a  magnet 
to  permit  the  air- 
stream  to  pass  into 
the  absorbing  tubes. 


26 


STUDIES  IN  PLANT  RESPIKATION  AND  PHOTOSYNTHESIS. 


in  the  mercury  in  the  small  bottle.  The  detail  in  figure  3  shows  what 
is  essentially  a  bell-valve  made  of  glass  and  sealed  with  mercury. 

As  is  shown  in  figure  2,  the  apparatus  contains  four  of  these 
valves,  each  with  its  own  electromagnet  and  each  connecting  with 
a  separate  absorption  tube.  The  time  during  which  each  valve 
was  held  open  represents  one  period  of  carbon-dioxid  determination. 

The  manner  in  which  the  electromagnets  are  controlled  can  be 
seen  by  referring  to  figure  4.     This  apparatus  consists  essentially 


Figure  4. 
The  clock  which  makes  electrical  contact  through  the  mercury  trough   and 
thus  controls  the  valves  by  means  of  the  electromagnets. 


of  a  circular  piece  of  wood  5  cm.  thick  and  30  cm.  in  diameter. 
A  circular  trough  has  been  cut  in  the  upper  face.  The  whole  is 
mounted  on  three  wooden  legs.  In  the  center  hole  is  a  24-hour 
cylinder^clock,  C.  This  carries  a  metal  arm,  on  one  end  of  which 
is  a  mercury  cup,*'and  through  this  cup  connection  is  made  with  the 
electric  current:  of  the^line  through  the  wire  and  binding  post  L. 
From  the  otherend  of  the  arm  is  suspended  a  looped  piece  of  copper 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS.  27 

wire.  This  copper  wire  floats  on  the  mercury  in  the  trough.  The 
sides  of  the  trough  and  the  rest  of  the  wood  were  covered  with  several 
coats  of  Bakelite  paint  to  insure  insulation.  The  trough  is  parti- 
tioned off  with  thin  pieces  of  mica,  dividing  the  whole  trough  into 
quadrants  which  are  completely  insulated  from  each  other.  If 
necessary  the  trough  can  be  divided  into  a  larger  nuniber  of  sections. 
The  mercury  in  each  section  (in  fig.  4  there  are  four)  is  connected 
by  means  of  a  short  wire  to  a  binding  post  V.  A  wire  from  each  one 
of  these  binding  posts  connects  to  one  of  the  electromagnetic  coils, 
the  other  end  of  the  coil  connecting  to  the  line.  Thus  the  circuit 
is  completed,  the  current  running  from  the  line  to  binding  post 
L,  in  turn  through  the  mercury  cup  on  the  clock,  the  arm  (not  through 
the  clock !) ,  through  a  portion  of  the  mercury  in  the  trough,  through 
a  sectional  binding  post  V,  to  a  coil,  and  back  to  the  line. 

As  the  clock  moves,  the  arm  is  carried  over  the  trough,  making 
contact  in  one  section  of  the  mercury.  When  the  arm  reaches  a 
mica  intersection  it  breaks  the  connection  with  one  section  and  makes 
contact  with  another.  Thus  the  current  is  broken  from  one  coil 
and  run  through  another  coil,  which  process  closes  one  valve  and 
opens  another.  In  this  manner  a  24-hour  clock  with  four  intersec- 
tions makes  possible  four  6-hour  periods.  The  periods  can  be  in- 
creased or  shortened  at  will  by  changing  the  number  of  intersections 
or  by  using  a  clock  of  different  rotation.  For  this  work  commercial 
110-volt  A.  C.  current  was  used;  the  coils  were  constructed  with  a 
resistance  of  about  80  ohms  and  heated  very  little.  Sparking  can 
be  avoided  by  insertion  in  the  circuit  of  suitable  condensers,  although 
no  trouble  was  experienced  from  this  source. 

From  the  mercury  valves  the  air-stream  passes  to  the  absorbing 
tubes.  These  were  10-bulb  Meyer  tubes  which  were  supported 
by  wooden  racks.  The  glass  tubes  were  connected  by  means  of 
heavy  rubber  tubing  and  all  rubber  connections  were  wired  and 
covered  with  Bakelite  paint.  Beyond  the  absorbing  tubes  a  pressure 
regulator  was  inserted.  This  was  patterned  after  the  well-known 
Palladin  regulator^  and  modified  so  that  a  cylinder  containing  a 
saturated  solution  of  calcium  chloride  was  used  as  the  liquid.  This 
permitted  more  accurate  adjustment  of  the  pressure  than  mercury. 
A  water  manometer  was  also  inserted  through  the  rubber  stopper 
in  the  cylinder,  so  that  any  change  in  the  pressure  in  the  system 
could  be  detected  at  once.  The  pressure  regulator  was  immersed 
in  the  water  of  the  thermostat  to  avoid  the  influence  of  changes  of 
temperature.  With  this  device  the  pressure  was  kept  quite  con- 
stant, never  exceeding  2  cm.  of  water,  and  the  air-stream  was  per- 
fectly regular.     Connection  was  made  from  the  pressure  regulator 

1  Abdebhalden,  E.     Handbuch  der  Biochemischen  Arbeitsmethoden,  vol.  iii,  1,  481  (1910). 


28  STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 

to  a  large  carboy  and  from  this  to  the  electric  pump.  The  rate  at 
which  air  is  drawn  through  the  apparatus  is  of  minor  importance 
as  long  as  it  allows  no  accumulation  of  carbon  dioxid  in  the  respiration 
chamber  and  as  long  as  the  carbon  dioxid  is  completely  absorbed 
in  the  barium-hydroxide  solution. 

The  carbon  dioxid  absorbed  by  the  barium-hydroxide  solutions 
was  calculated  from  determinations  of  the  changes  in  concentration 
of  this  solution.  At  first  this  was  done  by  titration  with  standard 
acid;  later  this  method  was  supplanted  by  the  more  rapid  and  more 
accurate  method  of  determining  the  electrolytic  conductivity  of  the 
solution. 

For  the  titration  method  a  solution  of  about  0.1  normal  strength 
was  used.  This  was  made  Up  in  18-liter  lots,  and  to  each  1,000  c.  c. 
1  gram  of  barium  chloride  was  added.  The  barium-hydroxide 
solutions,  through  which  carbon  dioxid  had  passed,  were  transferred 
to  narrow  bottles,  the  barium  carbonate  was  allowed  to  settle,  and 
three  aliquot  portions  of  the  clear  supernatant  solution  were  used  for 
titration  with  0.1  normal  hydrochloric  acid.  In  order  to  attain  very 
accurate  results  with  this  method  it  is  necessary  to  realize  that 
barium  carbonate  is  slightly  soluble  in  the  barium-hydroxide  solution. 
From  the  work  of  Vesterburg^  and  of  Weissenberger^  it  is  evident 
that  the  addition  of  barium  chloride  to  suppress  the  hydrolysis  of 
the  barium  carbonate  is  essential  in  order  to  attain  satisfactory 
results  with  this  method.  This  is  a  precaution  frequently  neglected 
in  determining  rates  of  respiration  by  this  procedure. 

On  account  of  the  labor  and  time  involved  in  making  the  many  ti- 
trations required  by  the  foregoing  method,  an  electrolytic  method 
was  devised  which  gave  exceedingly  satisfactory  results.  For  this 
purpose  a  pure  barium-hydroxide  solution  was  used,  prepared  with 
C02-free  water.  Considerable  preliminary  experimentation  was 
necessary  to  work  out  a  satisfactory  and  simple  apparatus  for  the 
electrolytic  determinations.  It  was  found  quite  impracticable  to 
incorporate  the  electrodes  for  the  conductivity  determinations  within 
the  absorption  tubes.  The  chief  difficulty  encountered  here  was, 
in  brief,  that  the  electrodes  became  covered  with  barium  carbonate 
and  thus  greatly  increased  the  resistance  of  the  cell.  Hence  the 
cell-constant  at  the  beginning  and  end  of  the  determination  was  not 
the  same;  even  a  very  slight  deposit  of  barium  carbonate,  such  as  is 
formed  by  wetting  the  electrodes  with  a  barium-hydroxide  solution 
and  allowing  this  to  remain  in  the  air  for  a  few  minutes,  gave  spurious 
results.  The  procedure  which  was  followed,  therefore,  was  to  transfer 
the  entire  contents  of  the  absorption  tube  into  a  glass  vessel  with  a 
wide  mouth.     This  was  carefully  stoppered  and  the  barium  car- 

>  Vesterburo,  A.     Zeit.  physik.  chem.,  70,  550-568  (1910). 
»  Weissenberger,  G.     Ibid.,  88,  257-270  (1914). 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


29 


bonate  allowed  to  settle  out  completely.  When  the  supernatant 
liquid  was  perfectly  clear,  this  vessel  was  placed  in  the  water  ther- 
mostat kept  at  25°.  The  stopper  was  replaced  by  a  dip-conductivity 
arrangement,  as  shown  in  figure  5.  The  electrodes  are  essentially 
the  same  as  those  used  in  the  Cain  method^  for  the  electrometric 
determination  of  carbon  in  steel.  A  Leeds  &  Northrup  combination 
bridge  and  galvanometer  operating  on  a  110-volt  A.  C.  60-cycle 
current  gave  highly  satisfactory  results. 
For  the  successful  operation  of  this 
method  a  number  of  jfacts  had  to  be 
determined  which  were  not  available  in 
satisfactory  form  in  the  chemical  liter- 
ature. It  was  necessary  to  choose  a 
barium-hydroxide  solution  of  a  con- 
centration such  as  would  completely 
absorb  the  carbon  dioxid  in  the  air- 
stream  at  the  maximum  rate  emitted 
by  the  leaves  and  for  the  longest  period 
of  observation.  Such  a  solution  should 
also  show  the  maximum  change  of  re- 
sistance for  a  given  change  in  concentra- 
tion. After  preliminary  experiments  in 
which  these  limits  were  established,  a 
solution  was  decided  upon  with  an  initial 
concentration  of  0.12  normal,  which 
could  wdth  safety  be  decreased,  through 
the   precipitation    of    barium    carbon- 

FlGUKE    5. 

Conductivity  cell  used  to  determine  the  strength  of 
the  barium-hydroxide  solution.  The  barium  carbon- 
ate is  allowed  to  settle  and  the  resistance  of  the  clear 
supernatant  solution  is  determined  at  25°. 


ate,  to  0.05  normal.  It  was  necessary  then  to  obtain  experimental 
data  from  which  a  curve  of  specific  resistance  could  be  drawn  for 
definite  concentrations  of  the  barium-hydroxide  solution.  The 
concentrations  of  the  solutions  used  for  these  determinations  were 
obtained  by  means  of  titration  with  hydrochloric  acid,  and  the 
resistances  were  determined  at  25°  by  means  of  the  cell  shown  in 
figure  5.  The  values  thus  obtained  are  given  in  table  7.  Fortu- 
nately there  were  available  two  determinations  of  the  specific  con- 
ductivity of  barium  hydroxide  by  A.  A.  Noyes^  at  concentrations 
very  close  to  both  extremes  of  the  concentrations  used  by  us.     Cal- 


1  Cain,  J.  R.,  and  L.,  C.  Maxwell.     Jour.  Ind.  and  Eng.  Chem.,  11,  852  (1919). 

2  NoYEs,  A.  A.     Tables  annuellea  constantes  et  donnees  num^riques  de  chimie,  de  physique  et 

de  technologie,  vol.  i,  p.  463  (1912). 


30 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


dilating  the  specific  resistance  from  these  values,  those  of  Noyes 
are  for  0.10  normal  48.97  and  for  0.05  normal  92.98,  while  our  results 
were  respectively  49.80  and  92.20. 

In  view  of  the  fact  that  our  determinations  were  not  made  for 
the  purpose  of  obtaining  physical-chemical  constants,  and  we 
required  only  relative  values  for  the  determination  of  respiration- 
rates,  the  agreement  is  quite  satisfactory. 

Table  7. — Specific  resistance  of  various  concentrations  of  barium-hydroxide  solution, 
determined  at  25°. 

Cell  constant  =  1 .2255. 


No. 

Normal 

Observed 

Specific 

No. 

Normal 

Observed 

Specific 

concentration. 

resistance. 

resistance. 

concentration. 

resistance. 

resistance. 

Stock 

0.11690 

53.2 

43.40 

6 

0.08297 

72.3 

59.00 

1 

0.11028 

55.8 

45.53 

7 

0.07771 

76.5 

62.42 

2 

0.10758 

57.0 

46.51 

8 
9 

0.06940 

84.6 

69.03 

3 

0.10337 

59.2 

48.30 

0.06111 

95.1 

77.60 

4 

0.09787 

62.4 

50.92 

10 

0.05717 

100.7 

82.17 

5 

0.08899 

67.8 

65.32 

11 

0.05221 

108.9 

88.86 

The  curve  plotted  from  these  determinations  is  given  in  figure  6. 
On  the  basis  of  these  values  it  was  possible  to  calculate  the  amount  of 
carbon  dioxid  represented  by  any  given  reduction  in  the  concen- 
tration of  the  absorbing  barium-hydroxide  solution.  The  manner 
in  which  this  was  employed  is  given  in  the  following  section. 

3.  The  Procedure  of  the  Respiration  Experiments. 

Before  using  the  apparatus  just  described,  it  was  of  course  tested 
for  small  leaks,  the  absorption  capacity  of  the  soda-lime  train  at  the 
required  rate  of  the  air-stream  was  determined,  and  the  limits  of 
complete  absorption  by  the  barium-hydroxide  solution  were  estab- 
lished. While  the  apparatus  seems  to  be  rather  complex,  for  con- 
tinuous use  for  an  extended  series  of  determinations  the  various 
appliances  proved  very  reliable  and  efficient. 

Before  each  experiment  the  respiration  chamber  was  washed  out 
with  a  solution  of  formaldehyde  and  then  with  distilled  water. 
Thereafter  filtered  air  was  drawn  through  the  chamber  until  the 
last  trace  of  formaldehyde  was  removed.  All  nutrient  solutions 
were  also  steriUzed  by  heating  twice  in  an  autoclave. 

Only  the  purest  chemicals  available  were  used.  The  sugars 
were  Pfanstiehl  brand.  Some  inconvenience  was  occasioned  by  the 
difficulty  of  procuring  reliable  amino-acid.  At  the  time  these 
experiments  were  begun  amino-acids  were  not  procurable  or  only  at 
an  exorbitant  price  and  of  a  quality  utterly  unfit  for  physiological 
work.     We  were  therefore  obliged  to  devote  about  three  months 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS.  31 

to  the  preparation  of  amino-acids.  The  laboratory  at  Carmel, 
California,  had  not  yet  been  completed,  and  we  are  greatly  indebted 
to  Professor  G.  N.  Lewis  for  extending  to  us  the  opportunity  of 
carrying  out  a  large  part  of  this  preparation  work  in  the  chemical 
laboratories  of  the  University  of  California.  We  were  thus  enabled 
to  prepare  adequate  quantities  of  a  variety  of  amino-acids  of  the 
highest  purity. 

The  mineral  nutrient  solution,  recommended  by  Dr.  B.  M.  Duggar, 
was  the  following:  Solution  No.  1:  3  grams  CaS04  in  3,000  c.  c. 
HaO.  Solution  No.  2:  6  grams  MgS047H20  in  1,000  c.  c.  H2O. 
Solution  No.  3:  3  grams  Merck's  ''soluble  ferric  phosphate"  in 
1,000  c.  c.  H2O.  Solution  No.  4:  12  grams  KCl  in  1,000  c.  c.  H2O. 
The  complete  nutrient  solution  was  made  by  mixing  in  the  following 
proportions:  30  c.  c.  No.  1;  10  c.  c.  No.  2;  10  c.  c.  No.  4;  diluted  to  200 
c.  c. ;  and  then  40  c.  c.  of  No.  3  was  added.  This  mixture  gave  excellent 
results.  A  number  of  other  commonly  used  nutrient  solutions 
employed  in  the  preliminary  experiments  were  unsatisfactory 
because  the  leaves,  after  being  in  the  dark  for  some  time,  were 
wilted  at  the  tips,  had  dark  spots,  appeared  rather  yellow,  or  showed 
other  abnormal  conditions. 

All  the  solutions  were  thoroughly  sterilized  before  using  by  twice 
heating  in  the  autoclave  to  15  pounds  pressure.  The  mineral 
nutrient,  sugar,  and  amino-acid  solutions  were  heated  separately 
before  mixing.  The  dish  and  the  glass  tubes  which  held  the  leaf 
petioles  were  also  sterilized. 

Great  care  was  exercised  in  selecting  the  leaves  in  order  to  use 
those  of  the  same  age  and  development.  Consideration  was  also 
given  to  the  conditions  to  which  the  plants  had  been  exposed  previous 
to  the  cutting  of  the  leaves,  viz,  temperature  and  illumination. 
Where  this  was  of  importance  the  leaves  were  taken  only  when  these 
conditions  were  as  nearly  the  same  as  can  be  obtained  in  a  green- 
house. The  petioles  of  the  leaves  were  cut  under  distilled  water. 
They  were  immediately  taken  to  the  laboratory  and  about  an  inch 
of  the  petiole  cut  off  under  water.  In  the  meantime  the  nutrient 
solution  had  been  prepared  and  placed  in  the  respiration  chamber. 
The  leaves  were  then  quickly  transferred  to  the  solution  in  the 
chamber,  care  being  taken  that  a  drop  of  water  adhered  to  the  end 
of  the  petiole.  The  leaves  were  placed  as  loosely  as  possible,  with 
the  petioles  well  submerged  in  the  nutrient  solution.  The  glass 
cover  was  then  placed  over  the  chamber  and  then  the  metal  cover. 
The  level  of  the  water  in  the  thermostat  was  raised  so  that  it  covered 
the  entire  chamber.  Before  cutting  the  leaves  the  absorption  tubes 
had  been  filled  and  connected,  so  that  as  soon  as  the  chamber  was 
closed  the  air-stream  could  be  started  through  the  apparatus.  For 
about  the  first  hour  no  account  was  kept  of  the  carbon-dioxid  emis- 


32  STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 

sion,  as  results  obtained  during  this  period  would  be  spurious,  owing 
to  the  carbon  dioxid  of  the  air  in  the  chamber,  etc.  Thereafter, 
however,  the  determinations  of  the  rates  appear  to  be  quite  concor- 
dant, depending  upon  nutritional  conditions,  and  no  evidence  of 
traumatic  effects  was  noticeable.  We  did  not  find  it  advisable  to 
run  these  experiments  over  100  hours,  as  the  danger  of  infection 
developing  after  this  time  became  great,  and  this  naturally  would 
introduce  serious  error. 

To  return  to  the  barium-hydroxide  solution:  125  c.  c.  of  this 
solution  was  introduced  into  each  absorption  tube  by  means  of  a 
pipette.  After  the  air-stream  had  passed  through  one  of  these 
tubes  for  a  definite  period,  it  was  disconnected  from  the  apparatus, 
and  the  solution,  containing  the  suspended  barium  carbonate,  was 
transferred  to  a  glass  vessel  (see  fig.  5)  and  tightly  stoppered.     By 


.16 

Figure  6. 
Specific-resistance  curve  of    barium  hydroxide  at  concentrations  from  0.05  to  0.12 
normal.     The  ordinate  represents  the  specific  resistance,  while  on  the  abscissa  the  CO2 
gram  equivalents  of  125  c.  c.  barium-hydroxide  solution  are  plotted  for  concentrations  of 
0.05  to  0.12  normal. 

rapid  manipulation  there  is  no  appreciable  error  introduced  from 
the  carbon  dioxid  of  the  air.  The  barium  carbonate  was  allowed 
to  settle  until  the  supernatant  liquid  was  perfectly  clear.  The 
vessel  was  placed  in  the  thermostat  kept  at  2.5°  and  the  resistance 
determined  by  means  of  the  apparatus  already  described.  At 
frequent  intervals  the  electrodes  were  dipped  in  dilute  hydrochloric 
acid  and  while  not  in  use  kept  in  distilled  water.  With  these  pre- 
cautions the  cell-constant  remained  unchanged  for  a  long  time. 

As  was  described  in  the  previous  section,  a  curve  was  drawn  with 
the  ordinate  as  specific  resistances  and  on  the  abscissa  the  normal 
concentration.     From   this  data  the  carbon-dioxid  equivalent  for 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS.  33 

125  c.  c.  of  solution  (the  amount  used  in  the  absorption  tubes)  can 
easily  be  calculated  and  used  as  the  abscissa  of  the  curve  just  men- 
tioned. Thus,  after  the  observed  resistance  value  of  a  barium- 
hydroxide  solution  had  been  converted  into  the  correct  value  of 
specific  resistance  by  correcting  for  the  cell-constant,  the  CO2 
equivalent  could  be  directly  read  from  the  chart.  The  difference 
between  this,  and  the  CO2  equivalent  of  the  original  solution  gives 
the  amount  of  carbon  dioxid  emitted  during  the  period  the  air  passed 
through  the  barium-hydroxide  solution.  This  chart  is  reproduced 
in  figure  6.  By  this  means  the  respiration  rates  were  determined 
with  very  satisfactory  accuracy  and  consumption  of  a  minimum 
amount  of  time. 

4.  The  Analyses. 

For  each  experiment  15  to  20  mature  leaves  were  cut.  One-half 
of  these  were  taken  for  immediate  analysis;  the  rest  of  the  leaves 
were  analyzed  after  the  respiration  experiment.  Both  sets  were  of 
course  treated  exactly  alike.  Dry-weight  determinations  were  made 
in  duplicate.  The  leaves  were  folded  up  and  placed  in  wide-mouth 
weighing  bottles,  the  covers  placed  on  the  bottles,  and  weighed. 
The  weighing  bottles,  with  the  covers  on,  were  then  placed  in  an 
electric  oven  at  98°  to  insure  rapid  kiUing.  After  about  30  minutes 
the  covers  were  removed  and  the  material  was  dried  at  the  same 
temperature  for  24  hours.  The  weighing  bottles  were  then  placed 
in  a  desiccator  and  weighed.  This  dry  material  was  then  ground  in 
an  agate  mortar  to  a  fine  powder,  after  which  it  was  ready  for  analysis. 

For  the  determination  of  the  total  sugars,  0.5  gram  of  the  leaf 
material  was  hydrolyzed  for  3  hours  in  25  c.  c.  of  1  per  cent  hydro- 
chloric acid,  filtered,  neutralized  with  sodium  bicarbonate  made  up 
to  100  c.  c,  and  the  sugars  determined  with  an  alkaline  copper- 
sulphate  solution  by  a  method  the  details  of  which  have  already 
been  described. ^  Although  this  method  entails  more  work  than  some 
of  the  other  methods  in  common  use,  it  was  found  that  these  pre- 
cautions were  justifiable  in  order  to  obtain  accurate  results  with 
plant  material. 

The  determination  of  total  sugars  seemed  to  yield  sufficient  infor- 
mation relative  to  the  carbohydrate-supplj^  of  the  leaf,  especially 
in  the  light  of  previous  investigations  which  showed  that  with  ample 
water-supply  the  carbohydrate  balance  was  in  favor  of  the  simpler 
sugars,  hexoses  and  disaccharides.  Special  experiments  showed 
that  the  method  of  hydrolysis  used  (1  per  cent  hydrochloric  acid) 
affected  only  the  reserve  starch  and  not  the  structural  elements,  the 
cellulose  of  the  leaf.  It  could  therefore  be  assumed  that  the  values 
for  the  total  sugars  give  an  indication  of  the  amount  of  carbohydrate 
material  which  was  available  to  the  leaf  for  respiratory  purposes. 

1  Spoehr,  H.  A.     Carnegie  Inst.  Wash.  Pub.  No.  287,  31  (1919). 


34  STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 

For  the  determination  of  amino-acids,  1  gram  of  the  dry  material 
was  heated  for  3  hours  with  25  c.  c.  of  water  on  the  boiling  water-bath. 
This  was  filtered  and  thoroughly  washed  with  hot  water.  The 
filtrate  and  washings  were  concentrated  on  the  water-bath  to  25  c.  c. 
It  was  found  inadvisable  to  try  to  remove  the  small  amount  of  soluble 
protein  from  this  extract,  because  any  procedure  designed  to  accom- 
plish this  also  affected  the  amino-acid  content,  and  this  amount  of 
protein  exerted  an  exceedingly  slight  influence  on  the  amino-acid 
determinations.  These  determinations  were  made  by  means  of 
the  Van  Slyke  micro  apparatus.  Deleano^  stated  that  the  nitrous- 
acid  method  of  determining  amino  groups  was  unreliable  when  there 
were  carbohydrates  and  nitrates  present  in  the  mixture.  Special 
tests  made  with  the  Van  Slyke  apparatus  with  pure  amino-acids  to 
which  nitrates  and  sugars  were  added  did  not  confirm  this  statement 
of  Deleano. '  With  some  of  the  plant  extracts  there  was  considerable 
frothing,  but  this  was  obviated  by  the  use  of  a  drop  of  caprylic 
alcohol  added  to  the  reaction  mixture  in  Van  Slyke  apparatus. 

The  results  of  the  respiration-rate  determinations  are  reported 
on  the  basis  of  the  amount  of  carbon  dioxid  emitted  per  gram 
of  dry  material.  This  is  of  course  an  arbitrary  method  and  of  only 
relative  value.  However,  there  exists  as  yet  no  rational  basis  in 
plant  physiology  of  connecting  the  respiratory  activity  of  vegetable 
organisms,  and  the  establishment  of  such  a  basis  must  await  a 
clearer  understanding  of  the  nature  of  the  process  itself.  The 
basis  of  fresh  weight  has  the  great  disadvantage  that  the  alterations 
in  water-content,  which  are  easily  effected  in  plants,  very  materially 
change  the  results.  The  use  of  a  surface  standard,  as  has  been  done 
in  some  work  in  animal  physiology,  has  Uttle  to  recommend  it  in 
work  with  plants. 

In  the  interpretation  of  analytical  data  obtained  from  living 
material  great  care  must  be  exercised  to  refrain  from  accepting  the 
apparent  results  as  the  true  state  of  affairs.  Very  rarely  is  a  single 
chemical  change  unaccompanied  by  other  changes  which  may  run  in 
the  same  or  opposite  directions.  Especially  is  this  difficulty  encoun- 
tered in  the  interpretation  of  analytical  data  of  leaves  calculated 
on  the  basis  of  percentage  of  original  dry  or  fresh  material.  Thus 
the  increase  in  one  component  very  often  simply  means  the  decrease 
of  another  component  which  goes  to  make  up  the  total  mass  of  the 
original  material. 

In  the  tabulation  of  the  experimental  data  some  liberty  was  also 
taken  in  expressing  the  time  in  decimals  of  hours  instead  of  in  hours 
and  minutes.  However,  this  method  facilitates  greatly  the  work 
of  calculating  and  avoids  a  common  source  of  error. 

J  Deleano,  N.     Jahrb.  wisa.  Bot.,  51,  552  (1921). 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS.  35 

EXPERIMENTAL  RESULTS. 

1.  The  Normal  Course  of  Respiration. 

It  has  been  known  for  a  long  time  that  as  the  supply  of  carbohy- 
drates in  a  plant  diminishes  the  rate  of  carbon-dioxid  emission  also 
decreases.  This  in  general  may  be  conceived  of  as  a  matter  of  mass 
action,  in  that  as  the  concentration  of  the  available  material  to  be 
oxidized  becomes  less  the  rate  of  the  reaction  is  decreased.  It  has 
also  been  recognized,  however,  that  these  relations  are  not  as  direct 
and  as  simple  as  was  supposed  at  first,  for  there  are  upper  and  lower 
limits  where  other  complications  or  limiting  factors  enter  and  where 
the  general  complex  of  reactions  takes  other  courses  and  makes  use 
of  different  material.^  But  in  order  to  form  some  conception  of  the 
behavior  of  chlorophyllous  leaves  apart  from  the  photosynthetic 
process  it  is  necessary  to  know  what  might  be  termed  the  normal 
course  of  respiration,  meaning  thereby  the  rate  at  which  the  stored 
material  is  converted  into  carbon  dioxid  and  water  with  the  liberation 
of  energy.  In  many  investigations  on  the  photosynthetic  process 
the  assumption  that  respiration  proceeds  at  the  same  rate  in  the 
light  as  in  the  dark  has  been  accepted  as  definitely  established,  or 
the  possibility  of  any  variation  of  this  factor  has  been  totally  neg- 
lected. In  a  practical  sense  we  are  interested  in  photosynthesis 
as  a  process  in  which  the  amount  of  energy  stored  exceeds  that 
liberated  by  the  organism.  Nevertheless,  if  we  are  to  formulate 
any  sort  of  conception  of  the  synthetic  processes  or  make  a  quanti- 
tative measure  thereof,  the  reverse  action,  going  on  simultaneously, 
can  not  be  neglected. 

No  conception  such  as  the  basal  metabolism  in  animals  has  been 
introduced  into  plant  physiology,  and,  on  account  of  fundamental 
differences  in  the  two  organisms,  it  is  improbable  that  such  relations 
can  be  worked  out.  But  the  normal  course  of  respiration,  although 
of  necessity  a  rather  arbitrary  value,  offers  a  base-line,  as  it  were, 
on  which  subsequent  respiratory  activity  can  in  a  sense  be  super- 
imposed. Another  feature  of  such  respiration  rates  is  that  with 
sufficiently  short  periods  and  accuracy  of  CO2  determinations  it 
becomes  evident  that  these  rates  show  irregularities  which  emanate 
from  the  internal  w^orkings  of  the  leaves  and  are  independent  of  any 
external  conditions.  We  shall  call  attention  to  one  such  irregularity 
in  the  form  of  a  sudden  rise  in  the  respiration  rate  after  the  leaves 
had  been  in  the  dark  for  about  40  hours. 

In  order  to  give  a  comparison  of  excised  leaves  with  the  normal 
plant,  the  rate  of  respiration  of  an  entire  plant  was  determined. 
As  the  analytical  data  in  table  13  indicate,  the  plant  has  in  the  stems 

>  Spoehh,  H.  a.     The  carbohydrate  economy  of  cacti.     Carnegie  Inst.  Wash.  Pub.  No.  287 
(1919). 


36 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


and  roots  a  large  reserve  of  carbohydrates  on  which  it  can  draw  for 
the  maintenance  of  its  respiratory  activity.  Any  changes  in  respira- 
tion due  to  carbohydrate  exhaustion  do  not,  therefore,  in  the  case 
of  the  attached  leaves,  appear  for  a  considerable  time,  while  in  the 
case  of  excised  leaves  such  changes  become  apparent  after  relatively 
short  periods  of  darkness.  For  this  experiment  an  entire  Helianthus 
annuus  plant  was  used.  The  plant  was  growing  in  soil  in  a  glass  jar. 
Before  the  respiration  rates  were  determined,  the  soil  was  covered 
with  tinfoil  and  the  edges  and  portions  around  the  stem  of  the  plant 
were  sealed  with  cocoa-butter.  The  experiment  was  allowed  to  run 
for  94.25  hours,  after  which  the  plant  was  still  in  perfectly  healthy 
and  vigorous  condition.  The  results  of  the  determination  are  given 
in  table  8. 


Table  8. — Rate  of  emission  of  CO2  by  a  small  potted  plant  of  Helianthus  annuus. 
Soil  covered  with  tinfoil  and  sealed  with  cocoa-butter.     CO2  absorbed  in  Ba(0H)2  solution, 
0.11835  normal,  125  c.  c.  of  which  haa  the  equivalent  of  0.3254  gram  CO2. 


No. 


Time. 


Observed 

Grams  CO2 

Mg.  CO2 

Total 

resist- 

equivalent 

Grams 

Mg.  CO2 

per  hour 

Hrs. 

hours. 

ance  m 

of  125  c.c. 

CO2  ab- 

per 

per  gram 

ohms. 

Ba(0H)2 
solution. 

sorbed. 

hour. 

dry 
weight. 

1.25 
3.00 

1.25 
4.25 

54.85 

0.3140 

0.0114 

3.80 

4.61 

6.5 

10.75 

56.6 

.3024 

.0230 

3.53 

4.19 

G.5 

17.25 

57.5 

.2970 

.0284 

4.37 

5.18 

5.0 

22.25 

56.6 

.3024 

.0230 

4.60 

5.46 

6.0 

28.25 

57.3 

.2982 

.0272 

4.53 

5.37 

6.5 

34.75 

58.0 

.2942 

.0312 

4.80 

5.69 

6.5 

41.25 

57.9 

.2950 

.0304 

4.67 

5.54 

5.0 

46.25 

56.9 

.3008 

.0246 

4.92 

5.84 

6.0 

52.25 

57.5 

.2970 

.0284 

4.73 

5.61 

6.5 

58.75 

57.8 

.2954 

.0300 

4.61 

5.47 

6.5 

65.25 

57.5 

.2970 

.0284 

4.36 

5.17 

5.0 

70.25 

56.3 

.3040 

.0214 

4.28 

5.08 

6.0 

76.25 

56.7 

.3016 

.0238 

3.96 

4.70 

6.5 

82.75 

57.1 

.2992 

.0262 

4.03 

4.78 

6.5 

89.25 

56.9 

.3004 

.0250 

3.84 

4.55 

5.0 

94.25 

55.9 

.3070 

.0184 

3.68 

4.36 

ll''45'°a.m.  to  1  p.m.  . 
1  p.  m.  to  4  p.m 

4  p.  m.  to  lOi'SO"  p.m 
10''30°>  p.m.  to  5  a.m .  . 

5  a.m.  to  10  a.m  .... 
10  a.m.  to  4  p.m 

4  p.m.  to  lO'^SO'"  p.m 
10''30™p.m.  to  5  a.m.  . 

5  a.m.  to  10  a.m 

10  a.m.  to  4  p.m 

4  p.m.  to  10''30'°  p.m 
itf'SO'^p.m.  to  5  a.m.  . 

5  a.m.  to  10  a.m 

10  a.m.  to  4  p.m 

4  p.m.  to  10*^30"  p.m 
10'>30'"  p.m.  to  5  a.m .  . 

5  a.m.  to  10  a.m 


These  results  are  graphically  represented  by  the  curve  in  figure  7. 
An  initial  drop  in  the  respiratory  rate  is  noticeable  here;  it  is  very 
much  more  prominent  in  the  experiments  with  excised  leaves. 
The  striking  feature  of  this  curve  is  that  during  about  the  first  48 
hours  there  is  a  gradual  increase  in  the  rate  of  respiration.  During 
all  this  time  the  available  supply  of  fuel  material  is  diminishing  and 
there  are  no  alterations  in  temperature  or  other  external  conditions. 
It  therefore  seems  justifiable  to  conclude  that  some  internal  factor 
comes  into  play  which  acts  in  a  stimulatory  manner.  From  the 
experiments  hereinafter  recorded  it  would  seem  that  the  amino-acids 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


37 


are  themselves  this  factor,  or  are  very  intimately  associated  therewith. 
The  amino-acids  exert  this  stimulating  action  on  the  respiratory 
activity;  they  behave  very  much  like  a  catalyst,  accelerating  the 
rate  of  carbohj^drate  catabolism-  and  resulting  in  an  increased  release 
of  energy  when  the  supply  of  material  is  decreasing.     Thus  the 


Si        GO        65~ 
FiGUHE    7. 

Rate  of  respiration  of  an  entire  plant  of  Helianthus  annuus.  Soil  covered  with  tinfoil 
and  sealed  with  cocoa-butter.  The  ordinate  represents  rng.  COo  per  hour  per  gram  dry 
material,  and  the  abscissa  the  time  in  hours. 

catabolic  activity  of  the  plant  tends  to  be  maintained,  though  the 
store  of  fuel  is  ebbing,  until  photosynthetic  activity  again  replenishes 
the  fuel  supply  and  the  light  diminishes  the  amino-acid  content. 
True  to  the  nature  of  catalyst,  the  amino-acids  do  not  yield  the 
energy,  but  when  the  concentration  of  the  subsirat  (the  carbohydrates) 
is  sufficiently  decreased,  the  rate  of  the  general  reaction  also  decreases. 
This  accounts  for  the  dropping  in  the  latter  portion  of  the  curve. 


Table  9. 


Dry 

weight. 

Total  sugars 
in  dry  material. 

Amino  nitrogen 
in  dry  material. 

Original  condition 

After  71.25  hours 

in  daik 

p.cl. 
10.59 

12.45 

p.  ct. 
5.93 

2.39 

p.cl. 
0.102 

0.380 

These  respiration  curves  of  necessity  show  in  but  a  gross  manner 
the  course  of  respiratory  activity.  They  represent  the  resultant 
of  forces.  The  reversal  of  the  rate  (a  drop  in  the  curve)  simply 
indicates  when  an  influence  opposed  to  the  stimulatory  one  is 
the  more  potent;  it  does  not  show  when  these  various  forces 
started.  As  the  analytical  data  given  in  tables  12  and  13  show, 
leaves  attached  to  the  plant  draw  heavily  upon  the  carbohydrate 


38 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


supply  in  the  rest  of  the  plant.  This  undoubtedly  accounts  for  the 
fact  that  the  plant  is  able  to  continue  its  initial  respiratory  rate  for 
so  long  a  time,  which  is  in  decided  contradistinction  to  the  behavior 
of  the  excised  leaves  when  these  are  not  suppHed  with  carbohydrates. 


Table  10. — Rate  of  CO2  emission  of  8  leaves  of  Helianthus  annmts 
Petioles  in  nutrient  solution  containing  no  organic  substances. 

at  U°. 

No. 

Time. 

Hours. 

Total 
hours. 

Cubic 
centimeters 
0.1/NHCl. 

Mg.  CO2 
per  hour. 

Mg.  CO2  per 
hour  per  gram 
dry  material. 

0 
1 
2 
3 
4 
5 
6 
7 
8 
9 

SMo-"  p.m.  to  4'>3(r  p.m 

4''30-"  p.m.  to  8''45»  p.m 

8''45'>"  p.m.  to  9''15"  a.m 

9''15™  a.m.  to  3  p.m 

0.75 
4.25 

12.50 
5.75 
6.25 

11.75 
6.75 
5.0 

12.25 
6.00 

0.75 
5.00 
17.50 
23.25 
29.50 
41.25 
48.00 
53.00 
65.25 
71  25 

27.40 
60.30 
20.80 
18.50 
30.80 
19.60 
13.15 
29.60 
14  65 

14.190 
11.088 
7.964 
6.512 
5.764 
6.380 
5.786 
5.224 
5.268 

3.337 
2.607 
1.873 
1.531 
1.355 
1.500 
1.360 
1.229 
1.239 

3  p.m.  to  9''15°'p.m 

9''15"'  p.m.  to  9  a.m 

9  a.m.  to  SHS""  p.m 

3''45'"  p.m.  to  8''45°'  p.m 

8^  45"  p.m.  to  9  a.m       

9  a.m.  to  3  p.m.  .. 

In  table  10  are  given  the  results  of  the  rate  of  carbon-dioxid  emis- 
sion from  sunflower  leaves  which  had  been  cut  from  the  plant. 
The  petioles  of  the  leaves  were  immersed  in  a  sterilized  nutrient 
solution  which  contained  only  inorganic  salts.  The  following  analyti- 
cal data  give  an  idea  of  the  changes  of  material  in  the  leaves  during 
the  period  of  respiration. 


Figure  8. 
Solid  line  represents  rate  of 
respiration  of  8  leaves  of  Heli- 
anthus  annuua  at  24°;  petioles 
in  a  nutrient  solution  contain- 
ing no  organic  substances. 
Values  taken  from  table  10. 
Broken  line  represents  rate  of 
respiration  of  15  leaves  of  Can- 
ada Wonder  bean;  petioles  in 
nutrient  solution  containing  no 
organic  substance.  Values  tak- 
en from  table  11.  The  ordinate 
gives  mg.  CO2  per  hour  per 
gram  dry  material,  the  abscissa 
the  time  in  hours. 


In  table  11  similar  results  are  given  for  excised  bean  leaves. 
From  the  curves  of  these  respiratory  rates,  given  in  figure  8,  it  is 
evident  that  in  the  excised  leaves  the  rate  of  CO2  emission  declines 
rapidly  when  the  only  supply  of  carbohydrates  is  the  material  stored 
in  the  leaf,  and  that  these  respiratory  rates  decrease  with  but  a  slight 
variation,  except  at  about  the  forty-eighth  hour.  These  graphs 
represent  the  normal  course  of  respiratory  activity  of  excised  leaves. 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


39 


The  course  of  the  changes  of  material  was  followed  by  placing 
five  vigorous  potted  Helianthus  plants  in  the  dark  at  20°  and  every 
24  hours  cutting  10  to  12  leaves,  which  were  then  analyzed.  After 
the  plants  had  been  in  the  dark  for  96  hours  they  were  again  placed 
in  sunlight  in  the  greenhouse  for  4  hours  and  another  sample  of 
leaves  analyzed.     The  results  are  given  in  table  12. 

Table  11. — Rate  of  CO2  emission  of  15  leaves  of  "Canada  Wonder'^  bean  at  S4°. 
Petioles  in  nutrient  solution  containing  no  organic  substances. 


Total 
hours. 

Cubic 

Mg.  CO2 

Mg.  CO2  per 

No. 

Time. 

Hours. 

centimeters 

per  hour. 

hour  per  gram 

0.1 /N  HCl. 

dry  material. 

1 

3''30"  p.m.  to  9''30"  p.m 

6 

6 

33.95 

12.40 

2.793 

2 

0^30^  p.m.  to  3''30™  a.m 

6 

12 

23.10 

8.40 

1.892 

3 

3''30"  a.m.  to  9''30°"  a.m 

6 

18 

25.10 

9.10 

2.049 

4 

9''30™  a.m.  to  3''30°'  p.m 

6 

24 

24.10 

8.70 

1.959 

5 

3''3CF>  p.m.  to  9''30'"  p.m 

6 

30 

22.60 

8.30 

1.869 

6 

9^30°^  p.m.  to  9''30™  a.m 

12 

42 

35.60 

6.50 

1.464 

7 

g'^SO™  a.m.  to  3''3Cr  p.m 

6 

48 

19.10 

7.00 

1.576 

8 

3''30™  p.m.  to  9'^30"'  p.m 

6 

54 

18.10 

6.63 

1.495 

9 

9''30'"  p.m.  to  9'>30"' a.m 

12 

66 

30.25 

5.50 

1.238 

The  analyses  given  in  table  12  indicate  a  gradual  depletion  of  the 
carbohydrates  available  to  the  leaves  when  the  plants  are  left  in  the 
dark.  This  depletion  can  not  be  made  up  entirely  by  drawing  upon 
the  reserve  material  in  other  parts  of  the  plant.  In  spite  of  this 
depletion  it  is  noteworthy,  as  has  been  pointed  out,  that  the  rate  of 
respiration  of  the  entire  plant  rises  and  then  decreases  slowly,  as  is 

Table  12. — Analysis  of  leaves  of  Helianthus  annuus,  after  exposure  to 
light,  then  in  dark  for  96  hours,  and  again  in  light  for  4  hours. 


Amino 

Total 

Leaf  samplea  taken. 

Dry  weight. 

nitrogen  in 

sugars  m 

dry  material. 

dry  material. 

p.ct. 

p.ct. 

p.ct. 

After  5  hours  sunlight .  . 

17.45 

0.230 

1.824 

24  hovu-s  in  dark 

16.61 

0.200 

0.983 

48  hours  in  dark 

15.15 

0.271 

0.736 

72  hours  in  dark 

15.47 

0.254 

0.921 

96  hours  in  dark 

15.21 

0.286 

0.777 

4  hours  in  sunlight .... 

16.52 

0.203 

1.373 

shown  in  figure  7.  After  4  hours  of  photosynthetic  work  the  leaves 
have  again  accumulated  considerable  sugar.  The  amino-acids, 
however,  increase  in  the  dark  and  after  subsequent  exposure  of  the 
plant  to  light  again  decrease. 

In  table  13  are  given  the  results  of  plants  similarly  treated  in 
which  the  leaves,  stems,  and  roots  were  analyzed  separately.     Two 


40 


STUDIES  IN  PLANT  EESPIRATION  AND  PHOTOSYNTHESIS. 


similar  Helianthus  plants  of  the  same  age  and  size,  growing  in  large 
pots,  were  used;  the  one  was  analyzed  at  once,  the  other  after  being 
in  the  dark  at  20°  for  96  hours. 

Table  13. — Analyses  of  Helianthus  annuus  plants,  leaves,  stems,  and  roots  separately  after 
exposure  to  light  for  5  hours  and  after  being  in  the  dark  for  96  hours. 


After  exposure  to  light 
for  5  hours. 

After  being  in  dark  for 
96  hours. 

Leaves. 

Stems. 

Roots. 

Leaves. 

Stems. 

Roots. 

Dry  weight 

Amino  nitrogen  in  dry  material .... 
Total  sugar  in  dry  material 

p.ct. 
18.10 
.219 
1.61 

p.  ct. 

20.24 

.101 

12.17 

p.ct. 

"    ^116 

5.28 

p.ct. 
15.93 
.294 
.81 

p.ct. 
17.70 

.075 
11.36 

p.ct. 

";i04' 
3.92 

The  analyses  in  table  13  show  a  general  decrease  in  the  carbo- 
hydrate-content of  the  plant  kept  in  the  dark.  Here  also  the  leaves 
of  the  plant  kept  in  the  dark  show  an  increase  in  amino-acids.  The 
stems  and  roots,  however,  show  a  very  slight  decrease  in  amino 
compounds. 

The  experiments  of  Schulze  and  Castoro^  on  the  accumulation  of 
amino-acids  have  already  been  mentioned.  In  this  work  the  seedlings 
were  kept  in  the  dark  for  2  weeks  and  the  amino-acid  content  in 
these  plants  was  very  much  higher  than  in  the  ones  exposed  to  the 

Table  14. — Amino  nitrogen  in  beans  (Canada 
Wonder)  sprouted  on  sawdust. 


No.  of 
hours. 

In  light. 

In  dark. 

Amino 
nitrogen. 

Dry 

weight. 

Amino 
nitrogen. 

Dry 
weight. 

0 

24 

72 

96 

144 

192 

p.ct. 
0.252 

p.ct. 

p.ct. 

0.252 
.146 
.274 
.399 
.372 
.403 

p.ct. 



41.40 
39.81 
36.42 
29.22 
26.04 

.357 
.343 

.368 
.411 

36.41 
35.90 

32.89 
30.13 

light.  During  this  length  of  time  the  seedlings  were  capable  of 
developing  well-formed  chlorophyllous  leaves.  We  carried  out 
comparative  experiments  in  which  periodic  analyses  were  made  on 
germinating  seeds,  in  the  light  and  in  the  dark,  for  a  shorter  time, 
the  seedlings  having  still  available  some  stored  proteinaceous  matter. 
In  these  experiments  no  differences  of  consequence  were  observable 
between  the  seedlings  grown  in  the  dark  and  those  grown  in  the  light. 

1  Schulze,  E.,  and  N.  Castobo.     Zeitschr.  physiol.  chem.,  38,  244  (1903);  49,  72  (1906). 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


41 


With  mature  excised  leaves  kept  in  the  dark  the  change  in  amino- 
acid  and  carbohydrate  content  naturally  becomes  much  more 
marked.  Thus  in  table  16  are  given  the  results  of  analyses  of  an 
experiment  in  which  40  similar  leaves  of  about  the  same  age  and  size 
were  cut  from  plants.  These  plants  had  been  exposed  to  sunUght 
in  the  greenhouse  for  about  9  hours.  The  cut  leaves  were  placed 
in  battery  jars,  with  petioles  in  nitrogen-free  nutrient  solution,  and 
kept  in  the  dark;  every  24  hours  8  leaves  were  removed  and  analyzed. 


Table  15. — Amino  nitrogen  in  beans  (Canada 
Wojider)  sprouted  on  filter-paper. 

No.  of 
hours. 

In  light. 

In  dark. 

Amino 
nitrogen. 

Dry 
weight. 

Amino 
nitrogen. 

Dry 
weight. 

0 

24 

48 

96 

120 

p.cl. 
0.252 

p.ct. 

p.ct. 

0.252 
.146 
.232 
.248 
.342 

p.ct. 

41.40 
39.43 
31.04 
30.59 

.195 
.264 
.373 

38.18 
32.07 
32.32 

The  results  given  in  table  16  show  a  decided  and  regular  decrease 
in  the  carbohydrate-content  of  the  leaves  kept  in  the  dark.  Also, 
the  amino-acids  in  these  leaves  increased  with  continued  time  in 
the  dark,  although  there  was  no  inorganic  nitrogen  given  in  the 
solution.  It  would  appear,  therefore,  that  there  is  a  continuous 
formation  of  amino-acids,  presumably  from  proteins,  and  that  under 
these  conditions  the  rate  of  protein  decomposition  exceeds  the  rate 
of  protein  synthesis,  with  the  result  that  the  splitting  products  in 
form  of  amino-acids  accumulate  in  the  leaves.  The  effect  of  this 
process  on  the  rate  of  respiration  and  its  relation  to  the  carbohydrate 
economy  of  the  plant  can  in  part  be  gathered  from  the  following 
experiments. 

In  table  16  were  also  given  the  results  of  analyses  of  leaves  kept 
in  the  dark  and  showing  the  gradual  depletion  of  the  carbohydrate 
material  used  by  the  plant  as  material  from  which  it  derives  its 
energy.  The  course  which  the  rate  of  respiration  follows  under 
these  circumstances  has  been  described  in  figure  8.  These  phenom- 
ena are  of  fundamental  importance  and  appear  relatively  simple 
and  well  known.  The  fate  and  behavior  of  the  proteins  under  Uke 
circumstances  are,  however,  quite  obscure.  Furthermore,  it  is 
impossible  and  irrational  to  try  to  follow  the  fate  of  these  substances 
in  plant  respiration  without  simultaneously  considering  the  car- 
bohydrate economy.     As  a  counterpart  to  the  experiment  sum- 


42 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


marized  in  table  16,  the  results  of  a  similar  experiment  are  given  in 
table  17,  in  which  the  petioles  of  the  leaves  are  placed,  instead  of  in 
nitrogen-free  nutrient  solution  only,  in  such  a  solution  containing 
also  7  per  cent  of  d-glucose.  Previous  to  cutting  the  leaves  the 
plants  had  been  in  the  sunlight  in  the  greenhouse  for  about  9  hours. 


Table  16. 


-Analysis  of  excised  leaves  of  Helianthus  annuus  kept  in  the  dark  ai 
20°,  with  petioles  in  nitrogen-free  nutrient  solution. 


No.  of  hours 

Amino 

Total 

leaveB  were 

Dry  weight. 

nitrogen  in 

sugars  m 

kept  in  dark. 

dry  material. 

dry  material. 

p.ct. 

p.et. 

p.ct. 

0 

13.48 

0.149 

2.49 

24 

10.76 

.187 

1.51 

48 

10.10 

.268 

0.90 

72 

13.46 

.272 

0.69 

96 

10.33 

.422 

0.44 

From  the  results  given  in  table  17  it  is  evident,  as  was  shown  many 
years  ago  by  Boehm,  that  when  excised  leaves  are  placed  with  the 
petioles  in  a  nitrogen-free  nutrient  solution  containing  7  per  cent 
d-glucose  the  sugar  is  taken  up  into  the  leaves  and  accumulates 
there.  As  will  be  seen  later  at  higher  temperatures,  the  sugar  under 
these  circumstances  accumulates  but  very  little,  if  at  all.     However, 

Table  17. — Analysis  of  excised  leaves  of  Helianthus  annuus 
kept  in  the  dark  at  20°,  tcith  petioles  in  nitrogen-free 
nutrient  solution  plus  7  per  cent  d-glucose. 


Number  of 

Amino 

Total 

hours  leaves 

Dry  weight. 

nitrogen  in 

sugars  in 

kept  in  dark. 

dry  material. 

dry  material. 

V.ct. 

p.ct. 

p.ct. 

0 

10.73 

0.164 

1.86 

24 

13.24 

.249 

1.83 

48 

13.12 

.176 

2.42 

72 

13.29 

.268 

2.75 

96 

12.69 

.183 

3.91 

when  there  is  thus  an  abundant  supply  of  carbohydrates,  the  amino- 
acids  increase  less  than  in  the  absence  of  sugar.  The  questions 
which  arise,  then,  are :  Do  the  carbohydrates  in  any  manner  influence 
amino-acid  formation,  and  do  the  amino-acids  affect  the  rate  of 
carbohydrate  consumption  by  the  leaf-cells?  Also,  what  is  the 
influence  of  light  and  dark  in  the  formation  of  amino-acids?  The 
experiments  hereinafter  described  may  throw  some  light  on  these 
questions. 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


43 


2.  GlycocolL 

That  amino-acids  accelerate  the  rate  of  carbohydrate  consumption 
in  the  leaves  becomes  evident  from  the  following  experiment:  A 
number  of  healthy  leaves  of  about  the  same  age  were  cut  from  the 
plants  in  the  manner  already  described  and  placed  in  battery  jars. 
In  one  set  the  petioles  were  put  in  100  c.  c.  nitrogen-free  nutrient 

Table  18. 


Leaves  in — 

Dry  weight. 

Amino 

nitrogen  in 

dry  material. 

Total 

sugars  in 

dry  material. 

Original  condition             

p.ct. 
14.36 
14.31 
11.36 

p.ct. 

0.089 
.238 
.293 

p.ct. 

11.48 
3.34 
2.75 

Nutrient  solution  only         

Nutrient  solution  plus  glycocoU. . 

solution  plus  100  c.  c.  of  water;  in  the  other  set  the  solution  consisted 
of  100  c.  c.  of  the  same  nutrient  solution  plus  a  solution  of  0.5  gram 
glycocoU  in  100  c.  c.  of  water.  These  solutions  were  steriHzed  as 
usual  and  the  experiments  carried  out  in  duplicate.  After  remaining 
in  the  dark  for  72  hours  at  20°,  all  the  leaves  were  removed  from  the 
jars  and  analyzed.     The  results  of  the  analysis  are  given  in  table  18. 

Table  19. — Rate  of  €0%  emission  of  8  leaves  of  Helianthus  annuris  at  24°. 
Petioles  in  nutrient  solution  containing  0.11  per  cent  of  glycocoU. 


No. 

0 
1 
2 
3 
4 
5 
6 

8 
9 
10 

Time. 

Hours. 

Total 
hours. 

Cubic 
centimeters 
0.1  N  HCl 

Mg.  CO2 
per  hour. 

Mg.  CO2  per 
hour  per  gram 
dry  material. 

4''30°^  p.m.  to  5  p.m 

5  p.m.  to  10''15™  p.m 

10''15™  p.m.  to  9  a.m 

0.50 
5.25 

10.75 
6.50 
5.25 

12.50 
6.00 
8.00 
9.75 
7.00 
5.25 

5.75 
16.50 
23.00 
28.25 
40.75 
46.75 
54.75 
64.50 
71.50 
76.75 

49.00 
80.10 
39.00 
26.50 
55.95 
26.35 
30.50 
36.30 
28.00 
20.35 

20.548 
16.412 
13.200 
11.088 
9.864 
9.658 
8.382 
8.184 
8.800 
8.492 

3.570 
2.852 
2.293 
1.926 
1.748 
1.678 
1.456 
1.422 
1.529 
1.478 

3''30™  p.m.  to  8M5"  p.m 

gh45m  p  jji_  to  9''15"  a.m 

g'^lS'"  a.m  to  SHS'^  p.m 

3''15'"  p.m.  to  lli'lS^p.m.... 
ll^'lS^'p.m.  to  9  a.m 

From  the  results  given  in  table  18  it  appears  that  the  leaves 
placed  in  nutrient  solution  gained  only  in  amino-acid  content  and 
that  the  leaves  placed  in  a  glycocoU  solution  showed  an  even  greater 
gain  in  amino-acids.  It  is  also  evident  that  the  leaves  fed  glycocoU 
consumed  more  carbohydrates  than  did  the  ones  which  had  been 
placed  in  nutrient  solution  only. 


44  STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 

In  tables  10  and  11  and  figure  8  are  given  the  results  of  what  was 
termed  the  normal  course  of  respiration.  This  represents  the  rate 
of  carbon-dioxid  emission  from  Helianthus  leaves  in  the  dark  at  24° 
in  nutrient  solution  containing  no  organic  material,  and  indicates 
the  rate  at  which  the  carbohydrates  in  the  leaves  are  consumed. 


40  45 

Figure  9. 
Rates  of  respiration  with  and  without  glycocoll.  The  broken  line  represents  the 
rate  of  respiration  of  8  leaves  of  Helianthus  annuus  at  24°;  petioles  in  a  nutrient  solu- 
tion containing  no  organic  substances.  Values  taken  from  table  10.  Solid  line  repre- 
sents the  rate  of  respiration  of  8  leaves  of  Helianthus  anmms  at  24°,  petioles  in  nutrient 
solution  containing  0.11  per  cent  of  glycocoll.  Values  taken  from  table  19.  The  ordi- 
nate represents  mg.  CO2  per  hour  per  gram  dry  material,  the  abscissa  the  time  in  hours. 

As  a  counterpart  to  this,  an  experiment  was  carried  out  in  which 
the  nutrient  solution  contained  an  amino-acid  in  order  to  determine 
w^hat  the  influence  of  this  is  on  the  rate  of  respiration.  The  Helian- 
thus leaves  were  taken  from  the  same  plant,  a  very  large  and  strong 
one,  and  were  treated  in  precisely  the  same  manner.  The  results 
of  this  respiration  experiment  are  given  in  table  19  and  in  figure  9, 
the  graphs  of  the  two  experiments,  the  one  with  and  the  other  without 
amino-acid. 

The  analytical  data  representing  the  original  condition  of  the 
leaves  and  that  at  the  end  of  the  experiment,  after  having  been 
in  the  dark  for  76.75  hours  at  24°,  with  the  petioles  of  the  leaves 
in  nutrient  solution  containing  0.11  per  cent  glycocoll,  are  given  in 
table  20. 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


45 


In  order  to  compare  the  respiratory  activity  of  the  two  sets  of 
leaves,  those  with  and  those  without  glycocoll  in  the  nutrient 
solution,  the  determinations  of  carbon  dioxid  are  summarized  in 
table  21. 

From  the  foregoing  it  is  evident  that  the  leaves  which  had  been 
fed  glycocoll  respired  more  actively  than  those  without  the  amino- 
acid.     On  the  basis  of  fresh  material,  the  former  produced  22.43 

Table  20. 


Dry  weight. 

Amino 

nitrogen  in 

dry  material. 

Total 

sugars  in 

dry  material. 

p.  ct. 
14.45 
13.12 

p.ct. 

0.100 

.459 

p.ct. 
9.28 
2.36 

After  76.75  hours  in  dark.  .  . 

per  cent  more  carbon  dioxid  than  the  latter,  and  on  the  basis  of 
dry  material  the  respective  difference  was  15.09  per  cent  in  favor 
of  the  leaves  given  glycocoll.  It  is  also  evident  that  the  leaves  which 
had  had  glycocoll  showed  a  much  greater  reduction  in  the  amount 
of  carbohydrates;  the  slight  difference  in  the  length  of  time  the  two 
experiments  ran  would  have  but  slight  effect  on  these  results.     It 

Table  21. 


Total  mg. 
CO2  formed. 

Total  mg.  CO2 
formed  per  gram 
fresh  material. 

Total  mg.  CO2 

formed  per  gram 

dry  material. 

Without  glycocoll. 
WithglycocoU.... 

549.56 
862.51 

16.082 
19.690 

129.20 
149.80 

might  be  argued  that  the  leaves  given  glycocoll  had  an  original 
higher  content  of  carbohydrates  and  that  this  might  account  for 
the  greater  respiratory  activity.  However,  it  has  been  our  experi- 
ence in  a  great  many  cases  that  within  certain  upper  and  lower 
limits  the  initial  carbohydrate-content  does  not  influence  the  rate 
of  respiration  under  controlled  conditions  of  temperature  and  water- 
supply.  Finally,  it  is  also  noticeable  that  the  leaves  given  glycocoll 
have  a  higher  amino-acid  content  at  the  end  of  the  experiment. 

3.  d-Glucose. 

From  the  foregoing  experiments  the  simple  facts  become  evident 
that  when  excised  leaves  are  kept  in  the  dark  at  25°  the  amino- 
acid  content  rises,  and  that  when  the  leaves  are  given   glycocoll 


46 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


this  is  taken  up  in  the  leaves  and  stimulates  the  rate  of  respiration 
and  carbohydrate  consumption.  However,  the  excised  leaves  have 
as  their  only  supply  of  carbohydrates  the  material  stored  in  the 
leaves,  which  is  depleted  in  a  relatively  short  time.  It  has  been 
shown,  though,  that  sugars  are  easily  taken  into  the  leaf  through 
the  petioles  from  a  nutrient  solution.  Under  these  circumstances 
the  respiratory  rate  exhibits  some  interesting  variations.  In  table  23 
are  given  the  results  on  an  experiment  in  which  the  excised  Helianthus 
leaves  were  fed  d-glucose,  so  that  the  carbohydrate-content  was 
practically  the  same  at  the  beginning  and  end  of  the  experiment. 
During  the  course  of  the  experiment,  80  hours  at  24°,  the  amino- 
acid  content  increased  about  threefold.  The  analytical  data  are 
given  in  table  22. 

Table  22. 


Dry  weight. 

Amino 

nitrogen  in 

dry  material. 

Total 

sugars  in 

dry  material. 

Initial  condition 

After  80  hours  in  dark. . 

p.ct. 
13.42 
12.02 

p.ct. 

0.101 

.312 

p.ct. 
7.55 
7.03 

The  determinations  of  the  rate  of  respiration  are  given  in  table  23 
and  figure  10.  These  show  that  although  the  leaves  were  constantly 
receiving  sugar,  the  rate  of  CO2  emission  decreased  regularly  during 
the  first  30  hours.  Thereafter  the  respiration  rate  increased  so 
that  after  80  hours  this  was  slightly  above  the  initial  rate.  It  should 
be  noted  that  at  this  time  the  carbohydrate-content  was  slightly 
below  the  initial  condition,  while  the  amino-acids  had  increased 
considerably. 

Table  23. — Rate  of  CO2  emission  of  9  leaves  of  Helianthus  annuus  at  24°- 
Petioles  in  nitrogen-free  nutrient  solution  containing  7  per  cent  of  d-glucose. 


No. 

Time. 

Hours. 

Total 
hours. 

Cubic 
centimeters. 
0.1/N  HCl. 

Mg.  CO2 
per  hour. 

Mg.  CO2  per 
hour  per  gram 
dry  material. 

0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 

12'»15°'  p.m.  to  2''30°  p.m 

2»>30°  p.m.  to  9''30"  p.m 

9''30°'p.m.  to  9  a.m 

9  a.m.  to  3  p.m 

2.25 
7.00 

11.50 
6.00 
6.00 

12.25 
6.50 
6.50 

11.00 
7.50 
4.25 

2.25 
9.25 
20.75 
26.75 
32.75 
45.00 
51.50 
58.00 
69.00 
76.50 
80.75 

43.10 
64.85 
32.05 
31.25 
71.25 
41.45 
39.65 
66.85 
48.40 
27.00 

13.530 
12.409 
11.748 
11.440 
12.804 
14.036 
13.420 
13.354 
14.190 
13.970 

3.083 
2.828 
2.677 
2.607 
2.918 
3.198 
3.058 
3.043 
3.234 
3.183 

3  p-ro-  tf>  9  p-Tn 

9  p.m.  to  9»'15°'  a.m 

9»'15'°  a.m.  to  3''45°' p.m 

3*'45»  p.m.  to  10''15'°  p.m 

10''15"  p.m.  to  9''15"'  a.m 

9''15"  a.m.  to  4''45"  p.m 

STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


47 


In  the  following  experiment  the  conditions  were  the  same  as  in 
the  previous  one,  with  the  exception  that  the  initial  carbohydrate- 
content  was  lower  and  the  leaves  showed  a  greater  gain  in  amino 
acids.     The  petioles  of  the  excised  leaves  were  also  placed  in  a  nitro- 


FlQUBB    10. 

Rate  of  respiration  of  9  leaves  of  Helianthus  annuus  at  24°;  petiole  in 
nitrogen-free  nutrient  solution  containing  7  per  cent  of  d-glucose,  from  table 
23.  The  ordinate  represents  mg.  CO2  per  hour  per  gram  dry  material,  the 
abscissa  the  time  in  hours. 


gen-free  nutrient  solution  containing  7  per  cent  of  d-glucose,  but 
the  leaves  also  showed  no  gain  in  carbohydrate-content.  There 
was,  however,  a  very  decided  increase  in  the  amino-acids  after 
94.25  hours  in  the  dark.  The  analytical  results  are  presented  in 
table  24  and  the  course  of  respiration  in  table  25  and  figure  11. 

Table  24. 


Dry  weight. 

Amino 

nitrogen  in 

dry  material. 

Total  carbo- 
hydrates in 
dry  material. 

Original  condition       

p.ct. 
14.19 
11.74 

p.ct. 

0.132 

.697 

p.ct. 
1.64 
1.14 

After  94.25  hours  in  dark.  .  . 

The  noteworthy  feature  of  the  foregoing  experiment  is  the  very 
marked  drop  in  the  respiration  rate  during  the  first  35  hours,  and 
thereafter  the  decided  increase,  so  that  the  rate  was  higher  at  the 
end  of  the  experiment  than  at  the  beginning.  From  previous  experi- 
ments, as  indicated  in  table  17,  it  seems  evident  that  when  leaves 
are  placed  with  the  petioles  in  a  7  per  cent  glucose  solution  the  carbo- 
hydrate-content is  maintained  after  the  first  24  hours,  so  that  it 
may  be  assumed  that  these  leaves,  with  their  initial  relatively  low 
carbohydrate-content,  did  not  suffer  a  decided  reduction  of  this 
material.  It  is  evident  that  after  about  35  hours  the  rate  of  CO2 
emission  increased  decidedly,  so  that  at  the  end  of  94.25  hours,  in 


48 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


spite  of  the  slightly  lower  carbohydrate-content,  the  leaves  showed 
a  higher  respiration  rate  than  at  the  beginning  of  the  experiment. 
This  indicates  that  after  about  35  hours  there  are  produced  within 
the  leaf  conditions  which  favor  a  more  active  catabolism  of  carbo- 
hydrates. From  the  experiments  already  described  and  others 
which  are  to  follow,  it  appears  certain  that,  when  leaves  are  kept  in 
the   dark,   synchronous  with  the  reduction  of  the  carbohydrates 

Table  25. — Rate  of  emission  of  CO2  by  6  leaves  of  Helianthus  annuus  at  25°. 

Petioles  in  nitrogen-free  nutrient  solution  containing  7  per  cent  d-glucose.     CO2  absorbed 
in  Ba(0H)2  solution  0.1169  normal,  125  c.c.  of  which  has  the  equivalent  of  0.3214  gram  CO2. 


No. 

Time. 

Hrs. 

Total 
hours. 

Observed 
resist- 
ance in 
ohms. 

Gram  CO, 
equivalent 
of  125  c.  c. 
Ba(0H)2 
solution. 

Gram 
CO2  ab- 
sorbed. 

Mg.  CO2 
per 
hour. 

Mg.  CO2 
per  hour 
per  gram 

dry 
weight. 

■ 

5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 

llM5"  a.m.  to  1  p.m..  . 

I  p.m.  to  4  p.m 

4  p.m.  to  10''30"  p.m .  . 
10^30°"  p.m.  to  5  a.m .  . 

5  a.m.  to  11  a.m 

II  a.m.  to  4  p.m 

4  p.m.  to  10''30»  p.m.  . 
10''30"  p.m.  to  5  a.m .  . 

5  a.m.  to  10  a.m 

10  a.m.  to  4  p.m 

4  p.m.  to  lO'^SO™  p.m.  . 
10''30"  p.m.  to  5  a.m. . . 

5  a.m.  to  10  a.m 

10  a.m.  to  4  p.m 

4  p.m.  to  10''30"  p.m. . . 
10''30™  p.m.  to  5  a.m. . . 

5  a.m.  to  10  a.m 

1.25 
3.00 
6.50 
6.50 
6.00 
5.00 
6.50 
6.50 
5.00 
6.00 
6.50 
6.50 
5.00 
6.00 
6.50 
6.50 
5.00 

1.25 
4.25 
10.75 
17.25 
23.25 
28.25 
34.75 
41.25 
46.25 
52.25 
58.75 
65.25 
70.25 
76.25 
82.75 
89.25 
94.25 

66.6 
83.1 
93.7 
69.8 
72.6 
74.9 
76.9 
71.5 
76.7 
77.3 
82.3 
77.4 
89.8 
99.6 

0.2522 
.1968 
.1730 
.2400 
.2298 
.2216 
.2154 
.2336 
.2160 
.2140 
.1988 
.2134 
.1812 
.1612 
.1340 
.1740 

0.0692 
.1246 
.1484 
.0814 
.0916 
.0998 
.1060 
.0878 
.1054 
.1074 
.1226 
.1080 
.1402 
.1602 
.1874 
.1474 

23.06 
19.16 
18.38 

is!  32 
15.35 
16.30 
17.56 
17.56 
16.52 
18.86 
21.60 
23.36 
24.64 
28.83 
29.48 

6.80 
5.65 
5.42 

5^40 
4.23 
4.81 
5.18 
5.18 
4.87 
5.56 
6.37 
6.89 
7.27 
8.50 
8.69 

93.1 

there  takes  place  an  increase  in  the  amino-acids.  Moreover,  an 
increase  in  the  amino-acids  stimulates  the  rate  of  carbohydrate 
catabolism.  Under  ordinary  circumstances,  when  the  stores  of 
carbohydrates  are  being  depleted  rapidly,  the  effect  of  the  natural 
increase  in  amino-acids  is  but  slightly  noticeable,  as  was  seen  in 
figure  8.  When,  however,  the  leaf  is  fed  sugar  and  the  amino-acids 
increase  appreciably,  the  result  is  a  decided  stimulation  of  CO2 
emission.  These  relations  are,  however,  of  a  more  complex  nature 
than  would  appear  at  first  glance.  They  of  course  involve  the 
metabolism  of  the  proteins  as  a  source  of  the  amino-acids,  and  the 
converse  question  arises  as  to  what  extent  the  formation  of  amino 
acids  from  proteins  is  affected  by  a  decreasing  supply  of  carbohy- 
drates.   These  questions  will  be  referred  to  later  in  this  paper. 

Before  taking  up  the  effect  of  amino-acids  on  respiration  in  more 
detail,  there  remains  to  be  described  the  course  of  respiration  under 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


49 


conditions  in  which  there  is  a  decided  increase  in  the  carbohydrate- 
content  of  the  leaves  and  but  a  shght  increase  in  amino-acids.  In 
the  following  experiment,  carried  out  with  Canada  Wonder  bean 
leaves,  the  experimental  conditions  were  the  same  as  in  the  two 


50   35^   40   4S    SO   5S   60   6S    70    75   80   85   90    95 

Figure  11. 
Rate  of  respiration  of  6  leaves  of  Helianthus  annuus  at  25°;  petioles  in  nitrogen- 
free  nutrient  solution  containing  7  per  cent  d-glucose.     The  ordinate  represents  mg. 
CO2  per  hour  per  gram  dry  material,  the  abscissa  the  time  in  hours. 

previous  experiments,  i.  e.,  the  petioles  of  the  leaves  were  in  a 
nitrogen-free  nutrient  solution  containing  7  per  cent  glucose,  and  the 
CO2  emission  was  determined  in  the  dark  at  25°.    From  the  analytical 

Table  26. 


Dry  weight. 

Amino 

nitrogen  in 

dry  material. 

Total 

sugars  in 

dry  material. 

p.ct. 
16.82 
14.70 

p.ct. 

0.335 

.469 

p.ct. 
2.53 
3.45 

After  58.35  hours  in  dark.  .  . 

data  in  table  26  it  is  evident  that  there  was  a  decided  increase  in 
the  carbohydrate-content,  but  only  a  slight  increase  in  the  amino 
acids.    The  rates  of  respiration  are  given  in  table  27  and  figure  12. 


50 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


It  should  be  noted  that  in  the  foregoing  experiment,  unlike  the 
two  previous  ones,  there  is  an  increase  in  the  carbohydrate-content 
and  the  increase  in  the  amino-acids  is  very  small.  With  these  facts 
in  mind,  the  results  of  the  rates  of  respiration  as  plotted  in  figure  12 
are  noteworthy.    The  rapid  drop  between  the  fifth  and  tenth  hours 

Table  27. — Rate  of  COi  emission  of  15  leaves  Canada  Wonder  hean  at  25°;   -petioles  in 
nitrogeiv-free  nutrient  solution  containing  7  per  cent  d-glucose. 
CO2  absorbed  in  Ba(0H)2  solution  0.1169  normal,  showing  resistance  of  53.2  ohms  in  cell  with 
constant  of  1.2255  at  25°. 


Total 
hours. 

Observed 

resist- 

Gram  CO2 
equivalent 

Gram 

Mg.  CO2 

Mg.  CO2 
per  hour 

No. 

Time. 

Hrs. 

of  125  c.  c. 

CO2  ab- 

per 

per  gram 

ohms. 

Ba(0H)2 

sorbed. 

hour. 

dry 

solution. 

weight. 

0 

1 

ll''55">a.m.  to  i^2'^  p.m.  . 
l'>2'"  p.m.  to  5''2°'  p.m 

1.10 
4.00 

1.10 
5.10 

68.3 

0.2422 

0.0792 

19.8 

5.13 

2 

5»'2'°  p.m.  to  ltf'32™  p.m. . 

5.50 

10.60 

71.7 

.2298 

.0916 

16.6 

4.30 

3 

10^32'°  p.m.  to3h32'"a.m. 

5.00 

15.60 

73.6 

.2232 

.0982 

19.6 

5.08 

4 

3»'32-"a.m.  to  8i'32°' a.m .  . 

5.00 

20.60 

68.1 

.2428 

.0786 

15.7 

4.07 

5 

8''32°'a.m.  to2''32°'p.m.. 

6.00 

26.60 

70.9 

.2328 

.0886 

14.7 

3.81 

6 

2''32°'p.m.  to  ll''17'"p.m. 

8.75 

35.35 

77.0 

.2120 

.1094 

12.5 

3.24 

7 

ll''17'»p.m.  to  4''17'°a.m. 

5.00 

40.35 

64.3 

.2592 

.0322 

12.4 

3.21 

8 

4''17'"a.m.  to  9''17'"a.m.  . 

5.00 

45.35 

67.6 

.2450 

.0764 

15.3 

3.97 

9 

9''17»a.m.  to  3'>17'"p.m.  . 

6.00 

51.35 

71.4 

.2310 

.0904 

15.1 

3.91 

10 

Si^lT""  p.m.  to  10''17'°p.m. 

7.00 

58.35 

71.0 

.2326 

.0888 

12.7 

3.29 

Figure  12. 
Rate  of  respiration  of  15 
leaves  of  Canada  Wonder  bean 
at  24°;  petioles  in  nitrogen-free 
solution  containing  7  per  cent 
d-glucose.  Values  taken  from 
table  27.  The  ordinate  repre- 
sents mg.  CO2  per  hour  per 
gram  dry  material,  the  abscissa 
the  time  in  hours. 


of  the  experiment  is  probably  due  to  the  fact  that  the  sugar  from 
the  nutrient  solution  had  not  yet  got  into  the  leaf  in  sufficient 
quantity  to  cover  the  loss  through  respiration.  Thereafter  the 
rate  of  CO2  emission  shows  a  rapid  rise,  soon  followed  by  a  regular 
decline  far  below  the  initial  rate,  until  the  fortieth  hour,  when  there 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


51 


again  occurs  the  characteristic  rise,  which,  however,  is  not  main- 
tained and  the  rate  again  falls,  so  that  after  about  60  hours  the  rate 
of  respiration  is  far  below  the  initial  rate,  in  spite  of  the  fact  that 
the  leaves  have  actually  increased  in  carbohydrate-content. 


Table  28. 


Dry  weight. 

Amino 

nitrogen  in 

dry  material. 

Total 

sugars  in 

dry  material. 

Initial  condition 

After  74  hours  in  dark. . 

p.ct. 
12.72 
12.78 

p.ct. 
0.124 

.888 

p.ct. 
1.33 
2.07 

Undoubtedly  the  carbohydrate  supply  alone  does  not  determine 
the  rate  of  respiration,  but  there  is  necessary  an  accessory  factor 
which  aids  the  successive  chemical  reactions  constituting  this  process. 
That  amino-acids  act  in  this  stimulating  manner  is  established  by 

Table  29. — Rate  of  emission  oj  CO2  by  6  leaves  of  Helianthus  annuus  at  25°. 
Petrioles  in  nitrogen-free  nutrient  solution  containing  7  per  cent  d-glucose  and  0.11  per   cent 
glycocoll.    CO2  absorbed  in  Ba(0H)2  solution  0.1169  normal,  125  c.  c.  of  which  has  the  equivalent 
of  0.3214  gram  CO2. 


No. 

Time. 

Hrs. 

Total 
hours. 

Observed 
resist- 
ance in 
ohms. 

Gram  CO2 
equivalent 
of  125  c.  c. 
Ba(0H)2 
solution. 

Gram 
CO2  ab- 
sorbed. 

Mg.  CO2 
per 
hour. 

Mg.  CO2 
per  hour 
per  gram 

dry 
weight. 

0 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

1.0 
2.0 
6.5 
6.5 
5.0 
5.0 
6.5 
6.5 
5.0 
2.0 
4.0 
6.5 
6.5 
5.0 
6.0 

1.0 
3.0 
9.5 
16.0 
21.0 
26.0 
32.5 
39.0 
44.0 
46.0 
50.0 
56.5 
63.0 
68.0 
74.0 

58.2 
74.6 
72.6 
70.8 
69.3 
77.2 
80.4 
72.2 
60.7 
68.0 
81.8 
84.6 
74.6 
81.4 

0.2930 
.2226 
.2294 
.2360 
.2412 
.2140 
.2040 
.2312 
.2798 
.2466 
.2000 
.1932 
.2228 
.2014 

0.0284 
.0988 
.0920 
.0854 
.0802 
.1074 
.1174 
.0902 
.0416 
.0748 
.1214 
.1282 
.0986 
.1200 

14.20 
15.20 
14.15 
17.08 
16.04 
16.52 
18.06 
18.82 

4.45 
4.77 
4.44 
5.36 
5.03 
5.18 
5.67 
5.90 

5  p.m.  to  lli'SO^^  p.m 

11''30"  p.m.  to  6  a.m 

6  a.m.  to  11  a.m         

11  a.m.  to  4  p.m 

4  p.m.  to  ICSO'"  p.m.... 
10''30"  p.m.  to  5  a.m 

5  a.m.  to  10  a.m         .... 

10  a.m.  to  12  m 

12  m.  to  4  p.m 

18.70 
18.60 
19.70 
19.70 
20.00 

5.87 
5.86 
6.19 
6.19 
6.28 

4  p.m.  to  \(^ZQi^  p.m 

10'>30'"  p.m.  to  5  a.m 

5  a.m.  to  10  a.m 

10  a.m.  to  4  p.m 

the  experiments  here  described,  and  the  point  naturally  suggests 
itself  that  under  normal  conditions  in  the  dark,  when  the  carbohy- 
drate-content decreases  and  the  amino-acids  increase,  there  is  a 
similar  stimulating  action  which  would  tend  to  maintain  a  relatively 
higher  respiration  rate.  From  such  data  as  are  available  it  appears 
that  the  accumulation  of  amino-acids  is  a  relatively  slow  process,  and 


52 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


it  seems  highly  probable  that  the  rise  in  the  respiration  rate  observed 
after  30  to  40  hours  represents  the  time  when  the  amino-acids  have 
accumulated  sufficientlj^  and  their  influence  on  the  rate  of  respiration 
becomes  noticeable.  The  experiments  on  this  point  will  be  taken 
up  later. 


40  50  60  70 

FlGtTBB   13. 

Rate  of  respiration  of  6  leaves  of  Helianthus  anmms  at  25°;  petioles  in  nitrogen-free 
nutrient  solution  containing  7  per  cent  d-glucose  and  0.11  per  cent  glycocoll.  Values 
taken  from  table  29.  The  ordinate  represents  mg.  CO2  per  hour  per  gram  dry  material, 
the  abscissa  the  time  in  hours. 

Turning  now  to  the  experiments  in  which  amino-acids  were  fed 
the  leaves,  it  should  be  noted  that  the  conditions  here  were  precisely 
the  same  as  in  the  foregoing  experiments,  with  the  exception  that 
the  nutrient  solution  contained,  besides  the  inorganic  salts  and  the 
specific  sugar,  a  definite  amount  of  amino-acid.     In  tables  23,  25, 

Table  30. 


Dry  weight. 

Amino 

nitrogen  in 

dry  material. 

Total 

sugars  in 

dry  material. 

p.ct. 
14.92 
12.71 

p.ct. 

0.142 

.653 

p.  ct. 
1.27 
1.07 

After  76.25  hours  in  dark .  .  . 

and  27  were  presented  the  results  of  respiration  determinations  of 
leaves  which  had  been  given  d-glucose.  In  the  following  experi- 
ment with  Helianthus  leaves  0.11  per  cent  of  glycocoll  was  added  to 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


53 


the  nutrient  solution.  The  analytical  data  show  that  during  the 
course  of  the  experiment  the  leaves  gained  both  in  amino-acids  and 
in  carbohydrate-content. 

Table  31. — Rate  of  emission  of  CO2  by  6  leaves  of  Helianthus  annuus  at  25°. 
Petioles  in  nitrogen-free  nutrient  solution  containing  7  per  cent  d-glucose  and  0.16  per  cent 
asparagine.     CO2  absorbed  in  Ba(0H)2  solution  0.11835  normal,  125  c.  c.  of  which  has  the  COj 
equivalent  of  0.3254  gram  CO2. 


No. 

Time. 

Hrs. 

Total 
hours. 

Observed 
resist- 
ance in 
ohms. 

Gram  CO? 
equivalent 
of  125  CO. 
Ba(0H)2 
solution. 

Gram 
CO2  ab- 
sorbed. 

Mg.  CO2 
per 
hour. 

Mg.  CO2 
per  hour 
per  gram 

dry 
weight. 

0 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

lli'45'"a.m.  to  1  p.m.... 
1  p.m.  to  4  p.m          .... 

1.25 

3.0 

6.5 

6.5 

5.0 

6.0 

6.5 

6.5 

5.0 

6.25 

6.5 

6.5 

4.75 

6.0 

1.25 
4.25 
10.75 
17.25 
22.25 
28.25 
34.75 
41.25 
46.25 
52.5 
59.0 
65.5 
70.25 
76.25 

60.7 
69.0 
71.4 
67.8 
72.2 
74.0 
75.3 
70.3 
80.2 
80.9 
82.4 
72.2 
81.5 

0.2800 
.2428 
.2340 
.2476 
.2312 
.2250 
.2202 
.2376 
.2046 
.2026 
.1986 
.2312 
.2010 

0.0454 
.0826 
.0914 
.0778 
.0942 
.1004 
.1052 
.0878 
.  1208 
.1228 
.1268 
.0942 
.1244 

15.13 

12.7 

14.06 

15.56 

15.70 

15.44 

16.18 

17.56 

19.32 

18.89 

19.50 

19.83 

20.73 

4.22 
3.54 
3.92 
4.34 
4.38 
4.30 
4.51 
4.90 
5.39 
5.27 
5.44 
5.53 
5.78 

4  p.m.  to  lO'^SO'"  p.m 

10i'30"' p.m.  to  5  a.m.... 

5  a.m.  to  10  a.m. 

10  a.m.  to  4  p.m 

4  p.m.  to  10'^30™  p.m 

IQi'SO™  p.m.  to  5  a.m 

10  a.m.  to  4''15"  p.m 

4''15"  p.m.  to  10*^45™  p.m. 
10'>45'»  p.m.  to  bHo""  a.m . 

5''15'»  a.m.  to  10  a.m 

10  a.m.  to  4  p.m 

From  the  determinations  of  the  rates  of  CO2  emission,  given  in 
table  29  and  figure  13,  it  is  apparent  that  when  the  leaves  are  given 
both  d-glucose  and  glycocoU  the  rate  of  respiration  rises  at  once 
and,  with  slight  irregularities,  maintains  this  increased  rate  for  at 
least  74  hours.  The  stimulating  action  of  glycocoll  becomes  evident 
when  the  graph  of  the  experiment  is  compared  with  the  three  pre- 
ceding ones.  This  is  especially  apparent  during  the  first  35  hours, 
that  is,  before  the  effect  of  the  amino-acids  accumulating  in  the 
leaves  becomes  noticeable. 

Table  32. 


Dry  weight. 

Amino 

nitrogen  in 

dry  material. 

Total 

sugars  in 

dry  material. 

Initial  condition 

After  76  hours  in  dark. . 

V.ct. 
15.33 
12.94 

V.ct. 

0.121 

.569 

V.ct. 
1.49 
1.03 

That  other  amino-acids  have  a  similar  effect  is  shown  by  the  fol- 
lowing experiment  with  asparagine  and  Helianthus  leaves.  All  con- 
ditions were  kept  precisely  the  same  and  the  nutrient  solution 
contained  0.16  per  cent  of  asparagine.    The  analytical  data  in  table  30 


54 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


show  that  while  the  initial  carbohydrate-content  was  not  quite 
maintained  at  the  end  of  the  experiment,  there  was  a  decided  gain 
in  amino-acids. 

The  determinations  of  the  rates  of  carbon-dioxid  emission  are 
given  in  table  31  and  figure  14.  A  noteworthy  feature  of  these 
results  is  the  rather  rapid  initial  drop  in  the  respiration  rate  before 
the  subsequent  rise.  This  is  probably  due  to  the  time  required  for 
the  asparagine  to  pass  through  the  petiole  and  penetrate  into  the 
cells.  GlycocoU,  on  the  other  hand,  has  notable  penetrating 
qualities,  and  the  effect  of  this  amino-acid  becomes  noticeable  very 
quickly  in  the  respiration-rate. 


20  30  40 

Figure  14. 
Broken  line  indicates  rate  of  respiration  of  6  leaves  of  Helianthus  annuus  at  25°; 
.  petioles  in  nitrogen-free  nutrient  solution  containing  7  per  cent  d-glucose  and  0.16  per 
cent  asparagine;  from  table  31.  The  solid  line  indicates  the  rate  of  respiration  of  leaves 
of  Helianthus  annuus  at  25°,  petioles  in  nitrogen-free  nutrient  solution  containing  7  per 
cent  d-glucose  and  0.11  per  cent  of  alanine;  from  table  33.  The  ordinate  represents 
mg.  CO2  emitted  per  hour  per  gram  dry  material,  the  abscissa  the  time  in  hours. 

Alanine  seems  to  be  taken  up  very  slowly.  The  same  conditions 
were  maintained  as  in  the  preceding  experiments  and  pure  alanine 
was  given  as  the  amino-acid.  The  analytical  data  in  table  32  show 
a  relatively  slight  increase  in  amino-acids  after  the  leaves  had  been 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


55 


in  the  dark  for  76  hours.  The  rate  of  respiration  as  shown  in  table  33 
and  figure  14  is  also  correspondingly  low  and  the  rise  in  the  rate 
comes  quite  late. 

Table  33. — Rate  of  emission  of  CO2  by  6  leaves  of  Helianthtis  annuus  at  25°. 
Petioles  in  nitrogen-free  nutrient  solution  containing  7  per  cent  glucose  and  0.11  per  cent 
alanine.    CO2  absorbed  in  Ba(0H)2  solution  0.11835  normal,  125  c.  c.  of  which  has  the  CO2  equiva- 
lent of  0.3254  gram  CO2. 


No. 

Time. 

Hrs. 

Total 
hours. 

Observed 
resist- 
ance in 
ohms. 

Gram  CO2 
equivalent 
of  125  c.  c. 
Ba(0H)2 
solution. 

Gram 
CO2  ab- 
sorbed. 

Mg.  CO2 
per 
hour. 

Mg.  CO2 
per  hour 
per  gram 

dry 
weight. 

0 

1 

\ 

4 
5 
6 
7 
8 
9 
10 
11 
12 
13 

1.0 
3.0 
6.5 
6.5 
5.0 
6.0 
6.5 
6.5 
5.0 
6.0 
6.5 
6.5 
5.0 
6.0 

1.0 
4.0 
10.5 
17.0 
22.0 
28.0 
34.5 
41.0 
46.0 
52.0 
58.5 
65.0 
70.0 
76.0 

1  p  m.  to  4  p.m 

65.5 
77.8 
77.9 
70.7 
72.5 
73.6 
73.9 
67.0 
78.9 
76.4 
77.2 
71.6 
78.3 

0.2570 
.2122 
.2118 
.2360 
.2300 
.2264 
.2252 
.2504 
.2086 
.2166 
.2140 
.2332 
.2106 

0.0684 
.1132 
.1136 
.0894 
.0954 
.0990 
.1002 
.0750 
.1168 
.1088 
.1114 
.0922 
.1148 

22.80 
17.41 
17.47 
17.88 
15.90 
15.23 
15.41 
17.43 

6.027 
4.602 
4.618 
4.726 
4.203 
4.026 
4.073 
4.607 

4  p.m.  to  10*'30™  p.m 

10''30™  p.m.  to  5  a.m 

10  a.m.  to  4  p.m 

4  p.m.  to  10''30°'  p.m 

10''30™  p.m.  to  5  a.m 

10  a.m.  to  4  p.m 

4  p.m.  to  10*'30"  p.m 

10''30"  p.m.  to  5  a.m 

5  H  Tn.  t-O  10  p  m  ,  . 

16.73 
17.13 
18.44 
19.13 

4.422 
4.528 
4.874 
6.056 

10  a.m.  to  4  p.m 

4.  Sucrose. 

Some  attention  was  also  given  to  the  influence  of  sugars  other 
than  d-glucose.  Thus  the  rate  of  respiration  was  determined  of 
leaves  which  were  given  sucrose  as  well  as  a  mixture  of  this  sugar 
and  an  amino-acid.  In  these  experiments  the  conditions  as  to 
temperature,  etc.,  were  the  same  as  in  the  preceding  determinations. 

Table  34  gives  the  analytical  data  of  the  leaves  in  the  initial 
condition  and  after  having  stood  in  the  dark  with  the  petioles  in  a 
nitrogen-free  nutrient  solution  containing  7  per  cent  of  sucrose. 

Table  34. 


Dry  weight. 

Amino 

nitrogen  in 

dry  material. 

Total 

sugars  in 

dry  material. 

p.ct. 
15.00 
14.00 

p.ct. 

0.126 

.335 

p.ct. 
2.14 

2.28 

After  76.5  hours  in  dark 

The  rates  of  respiration  of  this  experiment  are  given  in  table  35 
and  figure  15.  It  will  be  noted  that  although  there  is  a  slight  increase 
in  both  the  amino-acid  and  carbohydrate-content,  the  leaves  do 


56 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


not  maintain  the  initial  rate  of  CO2  emission  and  the  general  course 
of  the  rate  is  down.  The  rise  after  35  hours  is  very  noticeable;  this, 
however,  is  not  maintained  and  the  rate  gradually  drops. 

Table  35. — Rate  of  ennssion  of  CO2  by  6  leaves  of  Helianihus  annuus  at  25°. 
Petioles  in  nitrogen-free  nutrient  solution  containing  7  per  cent   sucrose.     CO2   absorbed   in 
Ba(0H)2  solution  0.11835  normal,  125  c.  c.  of  which  has  the  CO2  equivalent  of  0.3264  gram  CO2. 


No. 

Time. 

Hrs. 

Total 
hours. 

Observed 
resist- 
ance in 
ohms. 

Gram  CO2 
equivalent 
of  125  c.  c. 
Ba(0H)2 
solution. 

Gram 
CO2  ab- 
sorbed. 

Mg.  CO2 
per 
hour. 

Mg.  CO2 
per  hour 
per  gram 

dry 
weight. 

0 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

11''30'"  a.m.  to  12»'30™  p.m. 

12''30°  p.m.  to  4  p.m 

4  p.m.  to  10''30'"  p.m 

10''30"  p.m.  to  5  a.m .... 

1.0 
3.5 
6.5 
6.5 
5.0 
6.0 
6.5 
6.5 
5.0 
6.0 
6.5 
10.5 
1.5 
5.5 

1.0 
4.5 
11.0 
17.5 
22.5 
28.5 
35.0 
41.5 
46.5 
52.5 
59.0 
69.5 
71.0 
76.5 

64.6 
75.9 
76.1 
69.6 
73.6 
75.5 
78.9 
70.7 
75.4 
75.7 
102.7 
56.7 
72.6 

0.2608 
.2184 
.2172 
.2402 
.2264 
.2198 
.2088 
.2360 
.2198 
.2188 
.1554 
.3020 
.2298 

0.0646 
.1070 
.1082 
.0852 
.0990 
.1056 
.1166 
.0894 
.1056 
.1066 
.1700 
.0234 
.0956 

18.45 
16.46 
16.64 
17.04 
16.50 
16.24 
17.93 
17.88 
17.60 
16.40 
16.19 
15.60 
17.38 

5.42 
4.84 
4.89 
5.00 
4.84 
4.77 
5.26 
5.25 
5.17 
4.81 
4.75 
4.58 
5.10 

10  a.m.  to  4  p.m 

4  p.m.  to  10''30"  p.m .... 
10''30™  p.m.  to  5  a.m .... 

5  a.m.  to  10  a.m  . 

10  a.m.  to  4  p.m 

4  p.m.  to  lOHOP"  p.m 

10''30°'  p.m.  to  9  a.m 

9  a.m.  to   10'>30'"  a.m 

10''30°^  a.m.  to  4  p.m 

When,  on  the  other  hand,  the  leaves  are  given,  besides  sucrose, 
a  small  amount  of  glycocoU,  the  course  of  the  respiration  rates  is  a 
gradual  rise.  The  changes  in  material  are  given  in  the  analytical 
data  in  table  36. 


Table  36. 


Dry  weight. 

Amino 

nitrogen  in 

drj'  material. 

Total 

sugars  in 

dry  material. 

Initial  condition 

After  76  hours  in  dark. . 

V.ct. 
15.49 
14.11 

p.ct. 

0.086 

.310 

p.ct. 
2.11 
2.70 

The  rates  of  respiration  are  given  in  table  37  and  figure  15.  From 
these  it  is  apparent  that  after  an  initial  drop  in  the  rate  of  CO2 
emission,  which  probably  represents  the  time  required  for  the 
material  in  the  nutrient  solution  to  migrate  into  the  leaves,  there  is 
an  irregular  rise  which  becomes  pronounced  after  about  35  hours. 
The  principal  difference  between  the  leaves  which  have  been  given 
glycocoU  and  those  without  is  that  in  the  former  there  is  a  marked 
rise  in  the  rate  immediately  after  the  initial  drop.    This  is  probably 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS.  57 

attributable  to  the  influence  of  the  glycocoll  from  the  nutrient 
solution,  while  the  rise  after  35  hours,  which  is  noticeable  in  both 
experiments,  represents  the  effect  of  the  natural  accumulation  of 
amino-acids  in  the  leaves,  which  becomes  noticeable  even  in  those 
leaves  which  have  been  given  no  sugar  in  the  nutrient  solution. 


AO  50  60  70  90 

Figure  15. 
Rates  of  respiration  with  sucrose.  The  broken  line  indicates  the  rate  of  respiration 
of  6  leaves  of  Helianihiis  annuus  at  25°;  petioles  in  nitrogen-free  nutrient  solution  con- 
taining 7  per  cent  sucrose.  Values  taken  from  table  35.  The  soUd  line  indicates  the 
rate  of  respiraton  of  6  leaves  of  the  same  plant  at  25°;  petioles  in  nitrogen-free  nutrient 
solution  containing  7  per  cent  sucrose  and  0.11  per  cent  glycocoll.  Values  taken  from 
table  37.  The  ordinate  represents  mg.  CO2  per  hour  per  gram  dry  material,  the  abscissa 
time  in  hours. 

5.  d-levulose. 

As  was  pointed  out  in  the  introductory  discussion,  the  purely 
chemical  experiments  of  Nef,  as  well  as  the  physiological  studies  of 
Lusk,  point  to  the  conclusion  that  d-levulose  is  more  easily  oxidized 
than  any  of  the  other  hexose  sugars.  Among  plants,  particularly 
the  lower  ones,  there  is  a  great  diversity  in  the  capacity  for  using 
different  sugars.  There  exists  little  information  on  the  relative 
value  of  the  various  sugars  in  respect  to  respiratory  activity  of 
higher  plants.  Palladin^  attempted  to  determine  the  influence  of 
various  sugars  on  the  rate  of  respiration.  He  found  that  d-levulose 
produced  a  greater  carbon-dioxid  emission  than  either  d-glucose, 
sucrose,  maltose,  raffinose,  glycerine,  or  mannit.  However,  Palladin 
worked  with  etiolated  bean  shoots.  These  were  placed  in  solutions 
of  the  various  sugars  for  2  to  4  days  and  then  the  rate  of  respiration 

1  Palladin,  W.     Rev.  Gen.  de  BoL,  13,  19,  93,  127  (1901). 


58 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


was  determined  during  a  few  hours.  The  results  from  these  experi- 
ments can,  of  course,  not  be  taken  as  giving  definite  results  as  to 
the  use  in  respiration  of  the  various  sugars  employed.  First  of  all, 
no  account  is  taken  of  the  possible  conversion  of  the  sugars  in  the 
leaf  during  the  time  the  sprouts  were  in  the  sugar  solution.  Also, 
the  length  of  time  during  which  respiration  determinations  were 
made  was  entirely  too  short  to  gain  any  conclusive  idea  as  to  the 
true  rate  of  this  process,  and  it  is  very  questionable  whether  it  is 
justifiable  to  draw  conclusions  as  to  the  normal  behavior  of  plants 
from  a  study  of  etiblated  shoots.  Finally,  Palladin  carried  out  his 
respiration  determinations  with  levulose  in  diffuse  light,  which  is 
another  disturbing  factor. 

Such  evidence  as  is  now  available  seems  to  point  to  the  conclusion 
that  in  the  plant,  unlike  the  animal,  d-levulose  is  not  the  most 
easily  oxidized  sugar.  Brown  and  Morris^  made  comparative 
analyses  of  the  sugars  in  Troposolum  majus.  Two  sets  of  excised 
leaves  were  used;  the  one  was  analyzed  immediately,  the  other 
after  having  remained  in  the  dark  for  24  hours  with  the  petioles 
standing  in  water.    Thus  they  found: 

Table  37. — Rate  of  emission  of  CO2  by  6  leaves  of  Helianihus  annuus  at  25°. 
Petioles  in  nitrogen-free  nutrient  solution  containing  7  per  cent  sucrose   and  0.11  per  cent 
glycocoU.    CO2  absorbed  in  Ba(0H)2  solution  0.11835  normal,  125  c.  c.  of  which  has  the  equiva- 
lent of  0.3254  gram  CO2. 


No. 

Time. 

HrB. 

Total 
hours. 

Observed 
resist- 
ance in 
ohms. 

Gram  CO2 

equivalent 
of  125  c.  c. 
Ba(0H)2 
solution. 

Gram 
CO2  ab- 
sorbed. 

Mg.  CO2 
per 
hour. 

Mg.  CO2 

per  hour 
per  gram 

dry 
weight. 

0 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

1.0 
3.0 
6.5 
6.5 
5.0 
6.0 
6.5 
6.5 
5.0 
6.0 
6.5 
6.5 
5.0 
6.0 

1.0 
4.0 
10.5 
17.0 
22.0 
28.0 
34.5 
41.0 
46.0 
52.0 
58.5 
65.0 
70.0 
76.0 

1  p.m.  to  4  p.m 

59.6 
67.1 
69.8 
66.3 
70.1 
70.6 
71.5 
68.3 
73.8 
73.8 
74.7 
69.5 
73.9 

0.2856 
.2500 
.2400 
.2534 
.2384 
.2366 
.2334 
.2456 
.2256 
.2256 
.2224 
.2406 
.2250 

0.0398 
.0754 
.0854 
.0720 
.0870 
.0888 
.0920 
.0798 
.0998 
.0998 
.1030 
.1848 
.1004 

13.26 
11.60 
13.13 
14.40 
14.50 
13.66 
14.15 
15.96 
16.63 
15.35 
15.84 
16.96 
16.73 

3.84 
3.36 
3.80 
4.17 
4.20 
3.95 
4.10 
4.62 
4.81 
4.44 
4.58 
4.91 
4.84 

4  p.m.  to  lOi'SO"  p.m 

lO'^aO™  p.m.  to  5  a.m 

5am  to  10  a.m 

10  a.m.  to  4  p.m 

4  p.m.  to  lO'^SO™  p.m 

10'»30°  p.m.  to  5  a.m 

10  a.m.  to  4  p.m 

4  p.m.  to  10''30"  p.m 

10^30"  p.m.  to  5  a.m 

5  a.m.  to  10  a.m 

10  a.m.  to  4  p.m 

Evidently  there  is  a  considerable  loss  of  carbohydrates  due  to 
respiration,  as  well  as  a  decided  change  in  the  relative  amounts  of 
the  various  sugars.  In  all  probability  the  starch  was  converted  into 
maltose,  which  yields  dextrose,  and  the  cane  sugar  was  inverted  to 


>  Brown,  H.  T.,  and  G.  H.  Morris.    Jour.  Chem.  Soc.  London,  63,  671  (1893). 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


59 


equal  quantities  of  dextrose  and  levulose.  There  was  thus  un- 
doubtedly a  preponderance  of  dextrose.  Nevertheless,  there  was  a 
much  greater  increase  of  levulose  than  of  dextrose  during  the  24 


Table  38. 


Leaves  analyzed 
at  once. 

Leaves  kept  in 
dark  24  hours. 

Experiment  No .  . 
Starch 

III. 
3.693 
9.98 
0.00 
1.41 
2.25 

IV. 
5.425 
7.33 
0.00 
2.11 
2.71 

III. 

2.980 

3.49 

0.53 

3.46 

1.86 

IV. 
0.906 
3.35 
1.34 
3.76 
1.28 

Cane  sugar 

Dextrose 

Levulose 

Maltose 

hours  the  leaves  were  in  the  dark.  Brown  and  Morris  calculate 
from  these  results  that  far  more  dextrose  than  levulose  is  used  in 
the  respiratory  process,  as  is  shown  in  table  39. 

Table  39. — Stigars  used  up  in  respiration 
expressed  as  percentages  of  the  dry-leaf 
material. 


Expt.  III. 

Expt.  IV. 

Dextrose 

Levulose 

Maltose 

2.66 
1.19 
1.10 

0.65 
0.34 
5.94 

The  work  of  Parkin*  substantiates  the  conclusions  of  Brown  and 
Morris.  Working  with  Galanthus  nivalis,  the  leaves  of  which  do 
not  store  starch,  Parkin  found  that  levulose,  as  a  rule,  is  in  excess 
of  glucose,  irrespective  of  the  time  of  day  the  leaves  are  taken  for 
analysis. 

"Out  of  52  duplicate  leaf  analyses  made,  47  had  the  fructose  in  excess  of  the  glucose, 
and  only  7  the  reverse.  Representing  fructose  as  unity,  in  the  former  cases  the  ratio 
varied  from  1 :  0.4  to  1 :  0.76,  and  in  the  latter  from  1 : 1.01  to  1 : 1.06.  The  calculation 
of  the  separate  amounts  of  glucose  and  fructose  depends  chiefly  upon  one  observation, 
viz,  the  optical  angle  of  rotation.  A  slight  error  in  the  reading  of  this  will  affect 
the  results  considerably;  consequently  it  might  hardly  be  expected  that  any  further 
conclusion  beyond  the  bare  fact  of  the  excess  of  one  hexose  sugar  over  the  other 
could  be  reached." 

A  later  investigation  of  the  carbohydrates  of  foUage  leaves  by 
Gast^  yielded  very  similar  results.  Tropceolum  niojus  and  Vitis 
vinifera  during  24  hours  of  respiration  in  the  dark  showed  a  rela- 

1  Parkin,  J.     Biochem.  Jour.,  6,  1-47  (1911). 

2  Gast,  W.     ZeU.  physiol.  chem.,  99,  1-53  (1917). 


60 


STUDIES  IN  PLANT  EESPIRATION  AND  PHOTOSYNTHESIS. 


lively  greater  decrease  of  glucose  than  of  levulose.  While  Musa 
ensente  Gruel,  which  forms  very  little  starch,  showed  a  decrease  in 
cane  sugar  and  maltose  during  the  period  of  darkness,  glucose  in- 
creased and  levulose  remained  the  same.  The  results  with  Cucurbita 
ficifolia  Bet6  and  with  Canna  viridiflora  R.  and  Pav.  are  not  as  definite, 

Table  40. 


Dry  weight. 

Amino 

nitrogen  in 

dry  material. 

Total 

sugars  in 

dry  material. 

Initial  condition 

p.ct. 
16.21 
14.95 

p.ct. 

0.144 

.552 

p.ct. 
1.88 
1.60 

After  76.75  hours  in  dark .  .  . 

but  seem  also  to  indicate  that  in  the  course  of  respiration  more  of 
the  glucose  is  utilized.  The  expression  of  analytical  data  in  terms 
of  percentages  of  the  dry-leaf  material  makes  uncertain  the  calcula- 
tion of  the  amounts  of  the  various  sugars  used  up. 


Table  41. — Rate  of  emission  of  CO2  by  9  leaves  of  Helianihus  annuity  at  25°. 
Petioles  in  nitrogen-free  nutrient  solution  containing  7  per  cent  levulose.    CO2  absorbed   in 
Ba(0H)2  solution,  0.12153  normal,  125  c.  c.  of  which  has  CO2  equivalent  of  0.3343  gram  CO2. 


No. 

Time. 

Hrs. 

Total 
hours. 

Observed 
resist- 
ance in 
ohms. 

Gram  CO2 
equivalent 
of  125  c.  c. 
Ba(0H)2 
solution. 

Gram 
CO2  ab- 
sorbed. 

Mg.  CO2 

per 

hour. 

Mg.  CO2 
per  hour 
per  gram 

dry 
weight. 

0 

1 
2 
3 
4 
5 
6 
7 

I 

10 
11 
12 
13 

11^15^  a.m.  to  12i'15°>  p.m. 

12»'15°'  p.m.  to  4  p.m 

4  p.m.  to  10''30°^  p.m 

IQi^SO"  p.m.  to  5  a.m 

1.0 

3.75 

6.5 

6.5 

5.0 

6.0 

6.5 

6.5 

5.0 

6.0 

6.5 

6.5 

5.0 

6.0 

1.0 
4.75 
11.25 
17.75 
22.75 
28.75 
35.25 
41.75 
46.75 
52.75 
59.25 
65.75 
70.75 
76.75 

65.3 
74.1 
76.9 
68.2 
73.2 
74.0 
76.3 
69.0 
75.0 
75.6 
75.8 
68.3 
74.4 

0.2548 
.2214 
.2122 
.2426 
.2246 
.2218 
.2140 
.2396 
.2184 
.2164 
.2160 
.2422 
.2238 

0.0795 
.1129 
.1221 
.0917 
.1097 
.1125 
.1203 
.0947 
.1159 
.1179 
.1183 
.0921 
.1105 

21.20 
17.36 
18.78 
18.35 
18.30 
17.30 
18.50 
18.94 
19.31 
18.13 
18.20 
18.42 
18.41 

5.500 
4.503 
4.871 
4.760 
4.747 
4.487 
4.799 
4.913 
5.009 
4.703 
4.721 
4.778 
4.775 

10  a.m.  to  4  p.m 

4  p.m.  to  10^30°^  p.m 

10''30'°  p.m.  to  5  a.m.  .  . . 

5  a.m.  to  10  a.m 

10  a.m.  to  4  p.m 

4  p.m.  to  lO'^SO"  p.m.  .  .  . 
10''30°>  p.m.  to  5  a.m 

10  a.m.  to  4  p.m 

While  the  evidence  is  as  yet  by  no  means  conclusive,  it  appears 
that  levulose  in  leaves  is  more  stable  than  glucose.  The  question 
resolves  itself  into  whether  d-glucose  is  actually  more  easily  con- 
sumed in  the  leaf  or  whether  d-levulose  is,  under  certain  conditions, 
converted  into  d-glucose.  The  problem  of  the  glucose-fructose 
relation  is  a  very  fundamental  one  in  the  carbohydrate  economy  of 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


61 


plants.  Its  solution  is  associated  with  the  extraordinary  difficulty 
of  quantitatively  determining  the  various  sugars  in  leaves  which 
have  been  subjected  to  a  series  of  experimental  conditions. 


Table  42. 


Dry  weight. 

Amino 

nitrogen  in 

dry  material. 

Total 

sugars  in 

dry  material. 

Initial  condition 

After  76.25  hours  in  dark.  .  . 

p.ct. 
15.91 
13.85 

p.ct. 

0.190 

.615 

p.ct. 
1.969 
1.563 

Unfortunately  the  results  of  experiments  which  we  have  carried 
out  with  this  problem  in  view  had  to  be  discarded  on  account  of 
some  uncertainties  which  developed  later  in  our  analytical  methods. 
However,  the  investigation  of  this  phase  of  the  carbohydrate  econ- 
omy problem  is  to  be  continued. 

Table  43. — Rate  of  emission  of  CO2  hy  7  leaves  of  Helianihus  annuus  nl  25°. 
Petioles  in  nitrogen-free  nutrient  solution  containing  7  per  cent  d-levulose  and  0. 1 1  per  cent 
glycocoll.     CO2  absorbed  in  Ba(0H)2  solution,  0.11835  normal,  125  c.  c.  of  which  has  the  CO2 
equivalent  of  0.3254  gram  CO2. 


No. 

Time. 

Hrs. 

Total 
hours. 

Observed 
resist- 
ance in 
ohms. 

Gram  CO2 
equivalent 
of  125  c.  c. 
Ba(0H)2 
solution. 

Gram 
CO2  ab- 
sorbed. 

Mg.  CO2 
per 
hour. 

Mg.  CO2 
per  hour 
per  gram 

dry 
weight. 

0 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

11>'45"'  a.m.  to  1  p.m... 

.     1.25 
.    3.0 
.    6.5 
.    6.5 
.    5.0 
.    6.0 
.    6.5 
.    6.5 
.    5.0 
.    6.0 
.    6.5 
.    6.5 
.    5.0 
.    6.0 

1  25 

4.25 
10.75 
17.25 
22.25 
28.25 
34.75 
41.25 
46.25 
52.25 
58.75 
65.25 
70.25 
76.25 

64.4 
74.3 
76.6 
68.7 
71.7 
72.8 
74.0 
69.0 
73.9 
73.6 
74.3 
69.0 
74.2 

0.2620 
.2238 
.2160 
.2438 
.2332 
.2290 
.2246 
.2426 
.2250 
.2264 
.22.38 
.2426 
.2242 

0 . 0634 
.1016 
.1094 
.0816 
.0922 
.0964 
.1008 
.0828 
.1004 
.0990 
.1016 
.0828 
.1012 

21.13 
15.63 
16.83 
16.32 
15.36 
14.83 
15.50 
16.56 
16.73 
15.23 
15.63 
16.56 
16.86 

6.00 
4.44 
4.78 
4.64 
4.36 
4.21 
4.40 
4.71 
4.75 
4.33 
4.44 
4.71 
4.79 

4  p.m.  to  10^30™  p.m .  .  . 
10^30'"  p.m.  to  5  a.m.  .  . 

10  a.m.  to  4  p.m 

4  p.m.  to  10''30'°  p.m .  .  . 
10'»30"  p.m.  to  5  a.m .  .  . 

5  a.m.  to  10  a.m     .      ... 

10  a.m.  to  4  p.m 

4  p.m.  to  10'^30°'  p.m.  .  . 
10^30"'  p.m.  to  5  a.m... 

5  a.m.  to  10  a.m 

10  a.m.  to  4  p.m 

In  the  three  following  experiments  d-levulose  was  used  as  the 
sugar  in  the  nutrient  solution.  The  first  of  these  was  carried  out 
with  d-levulose  as  the  only  organic  substance  in  the  nutrient  solu- 
tion. The  analytical  data  of  the  changes  of  materials  in  the  leaves 
are  given  in  table  40. 

These  leaves  also  showed  the  usual  gain  in  amino-acids  after 
remaining  76.75  hours  in  the  dark.    There  was,  however,  a  decrease 


62 


STUDIES  IN  PLANT  RESPIBATION  AND  PHOTOSYNTHESIS. 


in  the  carbohydrate-content,  although  d-levulose  is  notably  a  good 
nutrient  for  starch-forming  plants.  The  rates  of  respiration  are 
given  in  table  41  and  figure  16. 


Table  44. 


Dry  weight. 

Amino 

nitrogen  in 

dry  material. 

Total 

sugars  in 

dry  material. 

Original  condition 

After  77  hours  in  dark. 

p.ct. 
18.31 
14.79 

p.  ct. 
0.157 

.480 

p.ct. 
2.16 
1.69 

The  preceding  experiment  was  repeated,  all  conditions  remaining 
the  same,  except  that  the  nutrient  solution  contained,  besides  the 
d-levulose,  0.11  per  cent  of  glycocoll.  The  analyses  of  the  leaves 
are  compiled  in  table  42. 

Table  45. — Rate  of  emission  of  CO2  hy  7  leaves  of  Helianthus  annuus  at  25°. 
Petioles  in  nitrogen-free  nutrient  solution  containing  7  per  cent  d-levulose  and  0.138  per  cent 
asparagine.     CO2  absorbed  in  Ba(0H)2  solution.     Nos.  1  to  8,  inclusive,  in  solution   0.11835 
normal,  125  c.  0.  of  which  has  the  CO2  equivalent  of  0.3254  gram  CO2.    Nos.  9  to  13  in  solution 
0.12153  normal,  125  c.  c.  of  which  has  the  CO2  equivalent  of  0.3343  gram,  CO2. 


No. 

Time. 

Hrs. 

Total 
hours. 

Observed 
resist- 
ance in 
ohms. 

Gram  CO2 
equivalent 
of  125  c.  c. 
Ba(0H)2 
solution. 

Gram 
CO2  ab- 
sorbed. 

Mg.  CO2 
per 
hour. 

Mg.  CO2 
per  hour 
per  gram 

dry 
weight. 

0 

1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 

lO'^SO™  a.m.  to  12  m 

1.5 
4.0 
6.5 
6.5 
5.0 
6.0 
6.5 
6.5 
5.0 
6.0 
6.5 
6.5 
5.0 
6.0 

1.5 
5.5 
12.0 
18.5 
23.5 
29.5 
36.0 
42.5 
47.0 
53.0 
59.5 
66.0 
71.0 
77.0 

73.0 
79.9 
81.5 
70.5 
74.5 
74.6 
73.9 
68.7 
70.5 
71.2 
73.1 
69.3 
76.0 

0.2282 
.2056 
.2010 
.2368 
.2232 
.2226 
.2250 
.2438 
.2366 
.2346 
.2280 
.2414 
.2182 

0.0972 
.1198 
.1244 
.0886 
.1022 
.1028 
.1004 
.0816 
.0977 
.0997 
.1063 
.0929 
.1161 

24.30 
18.43 
19.13 
17.72 
17.03 
15.81 
15.44 
16.32 
16.28 
15.33 
16.35 
18.58 
19.35 

5.521 

4.187 
4.346 
4.026 
3.869 
3.592 
3.508 
3.708 
3.700 
3.483 
3.715 
4.222 
4.396 

4  p.m.  to  10''30™  p.m.  .  .  . 
IQi'SO™  p.m.  to  5  a.m 

5  a.m.  to  10  a.m. .       .... 

10  a.m.  to  4  p.m 

4  p.m.  to  lO^ZQT  p.m 

10''30°^  p.m.  to  5  a.m 

10  a.m.  to  4  p.m 

4  p.m.  to  10'»30"  p.m 

10''30"  p.m.  to  5  a.m 

5  a.m.  to  10  a.m 

10  a.m.  to  4  p.m 

The  rates  of  respiration  for  this  experiment  are  given  in  table  43 
and  in  the  graphs  in  figur.e  16. 

The  experiment  was  again  repeated,  the  glycocoll  being  replaced 
in  this  instance  by  asparagine.  The  analytical  data  are  presented 
in  table  44. 

The  rates  of  respiration  are  shown  in  table  45  and  in  the  graphs 
in  figure  16. 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS.  63 

A  comparison  of  the  results  of  the  foregoing  three  experiments 
as  presented  in  jfigure  16  shows  at  once  that  apparently  d-levulose 
does  not  produce  an  increased  respiratory  activity  in  Helianthus 
leaves.  Also,  the  graphs  of  the  rates  of  respiration  differ  considera- 
bly from  the  experiments  in  which  d-glucose  and  sucrose  were  fed 
the  leaves.  Of  these  sugars,  d-levulose  seems  to  produce  the  least 
active  respiratory  activity  and  d-glucose  the  most  active,  while 
sucrose  lies  midway  between  the  two.     In  none  of  the  experiments 


J I L 


0  to  20  30  4C  SO  CO  70  80 

Figure  16. 
Rates  of  respiration  with  d-levulose.  The  dotted  line  indicates  the  rate  of  respira- 
tion of  leaves  of  Helianthus  annuus  at  25°;  petioles  in  a  nitrogen-free  nutrient  solution 
containing  7  per  cent  d-levulose;  values  from  table  41.  The  broken  line  indicates  the 
rate  of  respiration  at  25°  with  a  nitrogen-free  solution  containing  7  per  cent  d-levulose 
and  0.11  per  cent  glycocoU,  as  per  table  43.  The  soHd  line  indicates  the  rate  of  respi- 
ration at  25°  with  a  nitrogen-free  nutrient  solution  containing  7  per  cent  d-levulose  and 
0.138  per  cent  asparagine.  The  ordinates  represent  mg.  CO2  per  hour  per  gram  dry 
material ,  and  the  abscissa  the  time  in  hours. 

with  Helianthus  leaves  in  which  these  sugars  alone  were  given  was 
the  initial  sugar-content  maintained.  However,  the  reduction  in 
all  these  cases  was  about  the  same,  as  was  also  the  approximate 
increase  in  relative  water-content.  Nevertheless,  these  sugars  show 
decided  variation  in  their  effect  on  respiratory  activity.  Moreover, 
the  influence  of  adding  amino-acids  to  the  nutrient  solutions  con- 
taining either  d-glucose,  sucrose,  or  d-levulose  is  very  different. 
With  d-glucose  the  effect  is  marked,  particularly  before  the  fortieth 


64 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


hour  of  the  experiment  with  the  more  easily  penetrating  glj^cocoll 
and  asparagine.  With  d-levulose  no  stimulating  effect  can  be 
noticed  in  our  experiments;  in  fact  the  respiration  rates  in  the 
experiments  with  glycocoU  or  asparagine  and  d-levulose  are  relatively 
below  those  with  this  sugar  alone  during  the  first  half  of  the  time. 

6.  d-mannose. 

A  sugar  which  is  closely  related  in  structure  to  both  d-glucose 
and  d-levulose  is  d-mannose.  These  three  sugars  have  a  common 
enol  and  are  easily  converted  into  each  other.  ^  Aside  from  its 
rather  common  occurrence  as  an  anhydride,  mannan,  in  seed  coats 
and  similar  organs,  the  physiological  behavior  of  d-mannose  in 
plants  has  been  investigated  very  little.  Knudson^  found  that 
d-mannose  has  a  decidedly  toxic  action  on  the  root  tips  of  wheat 
and  peas  grown  in  sterile  agar  cultures. 

We  carried  out  but  two  complete  experiments  with  d-mannose, 
one  without  and  the  other  with  glycocoU  in  the  nutrient  solution. 
The  results  were  quite  surprising  in  that  there  was  no  toxic  action 
apparent  and  that  the  rates  of  respiration  developed  a  very  great 
increase  over  the  initial  rate. 

In  table  46  the  analytical  data  are  given  representing  the  changes 
in  material  in  leaves  which  have  been  given  d-mannose. 

Table  46. 


Dry  weight. 

Amino 

nitrogen  in 

dry  material. 

Total 

sugars  in 

dry  material. 

p.  a. 

14.68 
13.64 

p.ct. 
0.153 
0.788 

p.ct. 
1.24 
0.95 

After  77.5  hours  in  dark .... 

The  results  of  the  determination  of  respiration-rates  for  this  experi- 
ment are  given  in  table  47  and  figure  17. 

The  foregoing  experiment  was  repeated  with  the  addition  of  0.11 
per  cent  of  glycocoU  to  the  nutrient  solution.  The  analytical  data 
are  given  in  table  48. 

The  results  of  the  determination  of  the  respiration  rates  are  com- 
piled in  table  49  and  figure  17. 

The  experiments  with  d-mannose  are  noteworthy  on  account  of 
the  great  increase  in  amino-acids  in  both  cases.  Although  the 
petioles  of  the  leaves  were  standing  in  a  7  per  cent  solution  of 
d-mannose,  there  was  a  very  appreciable  decrease  in  the  total  sugar- 

1  Spoehh,  H.  a.     The  carbohydrate  economy  of  cacti.     Carnegie  Inst.  Wash.  Pub.  No.  287 

(1919). 

2  Knudbon,  L.     Amer.  Jour,  of  BoL,  4,  430-437  (1917). 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


65 


content  of  the  leaves.  Nevertheless,  the  respiration  rates  at  the 
end  of  the  experiment  show  a  remarkable  increase  over  the  initial 
rates,  although  the  latter  were  relatively  not  low.  There  is  here 
the  same  phenomenon,  exhibited  by  d-glucose  in  figure  11,  of  an 

Table  47. — Rate  of  emission  of  CO2  by  8  leaves  of  Helianthus  annum  at  25°. 
Petioles  in  nitrogan-free  nutrient  solution  containing  7  per  cent  mannose.     CO  2  absorbed 
in  Ba(0H)2  solution  0.12153  normal,  125  c.  c.  of  which  has  the  CO2  equivalent  of  0.3343  g.  CO2. 


No. 

Time. 

Hrs. 

Total 
hours. 

Observed 
resist- 
ance in 
ohms. 

Gram  CO2 
equivalent 
of  125  c.  c. 
Ba(0H)2 
solution. 

Gram 
CO2  ab- 
sorbed. 

Mg.  CO2 
per 
hour. 

Mg.  CO2 
per  hour 
per  gram 

dry 
weight. 

0 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

10*^30™  a.m.  to  12  m 

1.5 
4.0 
6.5 
6.5 
5.0 
6.0 
6.5 
6.5 
5.0 
6.0 
6.5 
6.5 
5.0 
6.0 

1.5 
5.5 
12.0 
18.5 
23.5 
29.5 
36.0 
42.5 
47.5 
53.5 
60.0 
66.5 
71.5 
77.5 

64.1 
72.7 
76.4 
68.1 
71.9 
75.1 
76.1 
71.0 
79.6 
83.2 
87.0 
79.3 
99.0 

0.2598 
.2262 
.2140 
.2428 
.2294 
.2180 
.2148 
.2326 
.2040 
.1944 
.1854 
.2048 
.1622 

0.0745 
.1081 
.1203 
.0915 
.1049 
.1163 
.1195 
.1017 
.1303 
.1399 
.1489 
.1295 
.1721 

18.62 
16.63 
18.50 
18.30 
17.48 
17.89 
18.38 
20.32 
21.71 
21.52 
22.90 
25.90 
28.08 

5.540 
4.947 
5.504 
5.444 
5.200 
5.322 
5.468 
6.045 
6.459 
6.402 
6.813 
7.706 
8.533 

4  p.m.  to  1U''30™  p.m 
10''30'"  p.m.  to  5  a.m 

4  p.m.  to  10^30"  p.m 
lO^'SO"  p.m.  to  5  a.m 

5  a.m.  to  10  a.m  .    . 

10  a.m.  to  4  p.m .... 

4  p.m.  to  10''30'"  p.m 
10''30™  p.m.  to  5  a.m 
5  a.m.  to  10  a.m 

10  a.m.  to  4  p.m .... 

increased  respiration  rate  with  decreased  total  sugar-content  and 
increased  amino-acids.  The  sugar-content  alone  is  no  index  of  the 
rate  at  which  the  leaves  utilize  this  material.  Moreover,  under  the 
conditions  of  the  experiment,  where  sugar  is  being  constantly  sup- 


Table  48. 


Dry  weight. 

Amino 

nitrogen  in 

dry  material. 

Total 

sugars  in 

dry  material. 

Initial  condition 

p.ct. 
15.00 
13.29 

p.ct. 
0.252 
0.741 

1.18 
0.72 

After  76.75  hours  in  dark .  .  . 

plied,  it  is  quite  conceivable  that  this  is  oxidized  as  rapidly  as  it  is 
taken  up.  This  state  of  affairs  must,  of  course,  also  frequently 
exist  under  natural  conditions.  But  the  increased  rate  of  respiration 
at  the  end  of  the  experiment  can  not  be  due  to  a  greater  supply  of 
sugar,  but  to  the  cooperation  of  some  other  factor  which  increases 
its  activity  with  time.  The  increase  in  amino-acids  under  the 
circumstances  is  more  than  a  coincidence  and  plays  an  important 


60 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


role  in  the  respiratory  activity.  In  the  experiments  with  d-mannose 
the  glycocoU  which  was  given  in  the  nutrient  solution  had  apparently 
very  little  effect,  as  the  normal  accumulation  of  amino-acids  in  the 
leaf  in  this  case  was  so  very  great.  There  must,  of  course,  also  be 
Umits  beyond  which  there  is  little  increase  in  amino-acids  and  above 
which  the  rate  of  respiration  does  not  rise,  so  that  further  addition 
of  an  amino-acid  would  be  of  little  consequence. 

Table  49. — Rate  of  emission  of  8  leaves  of  Helianthus  annuus  at  25°. 
Petioles  in  nitrogen-free  nutrient  solution  containing  7  per  cent  d-mannose  -f-  0.11  per  cent 
glycocoU.     CO2  absorbed  in  Ba(0H)2  solution  0.12153  normal,  125  c.  c.  of  which  has  the  CO2 
equivalent  of  0.3343  gram  CO2. 


No. 

Time. 

Hrs. 

Total 
hours. 

Observed 
resist- 
ance in 
ohms. 

Gram  CO2 
equivalent 
of  125  c.  c. 
Ba(0H)2 
solution. 

Gram 
CO2  ab- 
sorbed. 

Mg.  CO2 
per 
hour. 

Mg.  CO2 
per  hour 
per  gram 

dry 
weight. 

0 

1 

2 

3 

4 

6 

6 

7 

8 

9 

10 

11 

12 

13 

11>'15'"  a.m.  to  12i'15°>  p.m. 
12''15"  p.m.  to  4  p.m.  . .  . 

4  p.m.  to  10''30°'  p.m 

10*'30'"  p.m.  to  5  a.m  .... 

1.0 

3.75 

6.5 

6.5 

5.0 

6.0 

6.5 

6.5 

5.0 

6.0 

6.5 

6.6 

5.0 

6.0 

1.0 
4.75 
11.25 
17.75 
22.75 
28.75 
35.25 
41.75 
46.75 
52.75 
59.25 
65.75 
70.75 
76.75 

61.3 
67.1 
71.1 
66.3 
68.0 
70.0 
69.7 
65.2 
69.4 
72.0 
76.4 
73.0 
90.0 

0.2730 
.2468 
.2320 
.2493 
.2432 
.2358 
.2370 
.2552 
.2380 
.2288 
.2138 
,2254 
.1786 

0.0613 
.0875 
.1023 
.0850 
.0911 
.0985 
.0972 
.0791 
.0963 
.1055 
.1205 
.1089 
.1557 

16.34 
13.46 
15.73 
17.00 
15.18 
15.15 
15.00 
15.82 
16.05 
16.23 
18.53 
21.78 
25.95 

4.932 
4.036 
4.748 
5.131 
4.582 
4.573 
4.528 
4.775 
4.845 
4.899 
5.593 
6.574 
7.833 

10  a.m.  to  4  p.m 

4  p.m.  to  10''30°'  p.m 

10*^30"  p.m.  to  5  a.m 

5am  to  10  a  m 

10  a.m.  to  4  p.m 

4  p.m.  to  10''30'°  p.m 

10''30™  p.m.  to  5  a.m .... 

5  a.m.  to  10  a.m 

10  a.m.  to  4  p.m 

7.  Effect  of  the  Natural  Increase  in  Amino- Acids.     Influence  of  Light 
on  Amino-Acids  and  Effect  on  Respiration. 

In  the  experiments  which  have  been  described  attention  has 
repeatedly  been  called  to  the  rise  in  the  rates  of  respiration  after 
about  35  or  40  hours.  An  examination  of  the  graphs  shows  this 
rise  in  practically  all  of  the  experiments,  and  particularly  is  it 
noticeable  in  the  cases  in  which  no  amino-acid  was  contained  in 
the  solution.  It  is  perhaps  unnecessary  to  point  out  that  these 
graphs  do  not  represent  the  true  course  of  respiratory  activity,  for 
the  points  in  the  curves  represent  the  rates  of  carbon-dioxid  emission 
during  a  certain  period,  usually  6  hours.  Naturally  such  an  experi- 
mental procedure  makes  the  variations  appear  much  more  abrupt 
than  in  all  probability  they  are,  so  that  the  rise  in  respiration  rate 
between  the  thirty-fifth  and  fortieth  hour  must  be  regarded  with  the 
foregoing  in  view. 

When  excised  Helianthus  leaves  are  kept  in  the  dark,  with  the 
petioles  in  nitrogen-free  nutrient  solution  (table  16),  there  is  a  gradual 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


67 


rise  in  the  amino-acid  content  of  the  leaves.  Our  experiments  indi- 
cate that  amino-acids  have  a  stimulating  effect  on  the  respiratory- 
activity  of  leaves  containing  the  natural  sugars,  or  when  they  are  fed 
d-glucose  or  sucrose.  It  seems  permissible  to  assume  that  the  amino 
acids  which  accumulate  in  the  leaves  when  kept  in  the  dark  would 
exert  a  similar  stimulating  effect  if  the  leaves  contained  sufficient 


40  50  60  70  80 

Figure  17. 
Rates  of  respiration  with  d-mannose.  The  broken  line  indicates  the  rate  of  res- 
piration of  8  leaves  of  Helianthus  at  25°;  petioles  in  a  nitrogen-free  nutrient  solution 
containing  7  per  cent  d-mannose,  values  taken  from  table  47.  The  solid  line  indicates 
the  rate  of  respiration  of  8  leaves  of  Helianthus  at  25°  with  petioles  in  a  nitrogen-free 
nutrient  solution  containing  7  per  cent  d-mannose  and  0.11  per  cent  glycocoU,  values 
taken  from  table  49.  The  ordinate  represents  mg.  CO2  per  hour  per  gram  dry  material, 
the  abscissa  the  time  in  hours. 

sugar.  Normally,  of  course,  when  excised  leaves  are  kept  in  the 
dark,  besides  an  accumulation  of  amino-acids  there  is  a  rapid  reduc- 
tion of  the  sugar-content,  so  that  any  stimulating  effect  of  the 
accumulating  amino-acids  could  not  be  exerted  on  account  of  the 
reduced  fuel  material. 

If,  however,  after  the  leaves  have  remained  in  the  dark  for  a 
time  and  the  amino-acids  have  accumulated,  glucose  is  then  fed,  the 


68 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


effect  of  this  increased  amino-acid  content  becomes  noticeable  at 
once.  Thus  excised  leaves  which  had  been  kept  in  the  dark  for  about 
40  hours  and  then  given  d-glucose  exhibited  an  immediate  increase 
in  the  rate  of  carbon-dioxid  emission.  This  is  not  the  case  when 
leaves  are  given  d-glucose  immediately  after  cutting  from  the  plant; 
there  is  then  a  primary  drop  in  the  CO2  curve  and  a  rise  after  about  40 
hours  (see  figs.  10  and  11).  The  combination,  then,  of  the  accumu- 
lated amino-acids  after  40  hours  of  darkness  and  an  abundant  sugar 
supply  results  in  an  accelerated  rate  of  respiration. 

Table  50. — Rate  of  CO2  emission,  at  24°,  of  8  leaves  of  Helianthus  annuus. 
The  excised  leaves  were  placed  in  a  nutrient  solution  free  from  organic  naaterial  and  nitrogen 


and  kept  in  the  dark  for  43.5  hours.     They  were  then  transferred  to  a  nitrogen-free  nutrient 
solution  containing  7  per  cent  d-glucose  and  the  rates  of  respiration  determined. 

No. 

Time. 

Hours. 

Total 
hours. 

Cubic 
centimeters 
0.1/NHCl. 

Mg.  COo 
per  hour. 

Mg.  CO,  per 
hour  per  gram 
diy  material. 

1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 

11''30'"  a.m.  to  12''12"  p.m. . 

12''15'"  p.m.  to  5  p.m 

5  p.m.  to  8''3CF'  p.m 

8^30"^  p.m.  to  9  a.m 

0.75 
4.75 
3.50 

12.50 
6.00 
6.75 

11.75 
6.75 
5.50 

11.50 
6.00 

0.75 
5.50 
9.00 
21.50 
27.50 
34.25 
46.00 
52.75 
58.25 
69.75 
75.75 

27.65 
22.25 
84.50 
51.00 
56.20 
85.80 
54.65 
43.20 
87.85 
56.45 

12.80 
13.99 
14.85 
18.70 
18.26 
16.19 
17.60 
17.18 
17.01 
20.68 

2.387 
2.608 
2.768 
3.486 
3.404 
3.018 
3.283 
3.203 
3.171 
3.855 

3  p.m.  to  9^45'"  p.m 

9''45-"  p.m.  to  9^30™  a.m 

9''30™  a.m.  to  4^15°"  p.m 

4'>15°'  p.m.  to  9'^45'"  p.m 

gMS-"  p.m.  to  9''15'"  a.m 

gMS'^a.m.  to3''15«'p.m 

This  fact  becomes  evident  from  the  following  experiment,  in  which 
8  Helianthus  leaves  were  cut  from  the  plant,  placed  immediately 
in  nitrogen-free  mineral  nutrient  solution,  and  kept  in  the  dark. 
After  43.5  hours  the  leaves  were  put  in  the  respiration  chamber 
with  fresh  nutrient  solution  containing  7  per  cent  of  d-glucose. 
From  the  results  of  the  determinations  of  the  rates  of  carbon- 
dioxid  emission  in  table  50  and  figure  18  it  is  apparent  that  the  rate 
of  respiration  rises  immediately  and,  with  some  irregularities,  then 
attains  its  maximum. 

It  is,  moreover,  a  noteworthy  fact  that  when  the  foregoing  experi- 
ment is  repeated  in  such  a  manner  that  the  nutrient  solution  con- 
tains, besides  d-glucose,  0.11  per  cent  of  glycocoll,  there  is  practically 
no  difference  in  the  rate  of  carbon-dioxid  emission.  In  other  words, 
it  appears  that  the  natural  accumulation  of  amino-acids  is  just  as 
effective  in  stimulating  respiratory  activity  as  when  amino-acids 
are  fed  to  the  leaves.  It  was  shown  in  the  previous  experiments 
that  the  influence  of  amino-acids  when  fed  to  the  leaves  was  espe- 
cially noticeable  in  the  first  40  hours  of  the  experiment,  that  is,  before 
the  natural  accumulation  of  amino-acids  in  the  dark  becomes  eft"ective. 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


69 


It  might  be  argued  that  this  rapid  increase  in  the  rate  of  respira- 
tion of  leaves,  which  had  been  previously  left  in  the  dark  and  then 
given  d-glucose,  was  due  simply  to  the  greater  available  supply  of 

Table  51. — Rate  of  CO2  einission,  at  S4°,  of  S  leaves  of  Helianlhus  annuus. 
The  excised  leaves  were  placed  in  a  nutrient  solution  free  from  organic  material  and  nitrogen 
and  kept  in  the  dark  for  42.75  hours.     They  were  then  transferred  to  a  nutiient  solution  con- 
taining 7  per  cent  d-glucose  and  0.11  per  cent  glycocoll  and  the  rates  of  respiration  determined. 


No. 

Time. 

Hours. 

Total 
hours. 

Cubic 
centimeters 
0.1/N  NCI. 

Mg.  CO2 
per  hour. 

Mg.  CO2  per 
hour  per  gram 
dry  material. 

1 

2 
3 
4 
5 
6 
7 
8 
9 
10 

11''45"'  a.m.  to  12''30"'  p.m.  .  . 

12''30'"  p.m.  to  5  p.m 

5  p.m.  to  9*'45™  p.m 

QMS'"  p.m.  to  11  a.m 

11  a.m.  to  9''30'"  p.m 

Qi'SO""  p.m.  to  9  a.m 

9  a.m.  to  3''30™  p.m 

3*'30'°  p.m.  to  ll'>30"'  p.m.  .  . 

ll''30'°  p.m.  to  9'' IS'"  a.m 

^HS^  a.m.  to  3''15'"  p.m 

0.75 
4.50 
4.75 
13.25 
10.50 
11.50 
6.50 
8.00 
9.75 
6.00 

0.75 
5.25 
10.00 
23.25 
33.75 
45.25 
51.75 
59.75 
69.50 
75.50 

27.60 
34.10 
96.00 
92.30 
87.40 
58.40 
68.45 
85.55 
61.25 

13.49 
15.80 
15.97 
19.36 
17.63 
19.76 
18.81 
19.34 
22.44 

2.389 
2.798 
2.829 
3.429 
3.087 
3.500 
3. 332 
3.408 
3.975 

sugar.  In  the  experiments  already  described  it  has  been  pointed 
out  that  the  rate  of  respiration  bears  no  direct  relation  to  the 
carbohydrate-supply.  There  are  numerous  instances  in  which  an 
increase  of  carbon-dioxid  emission  was  obtained  with  a  decreased 
total  sugar-content.    In  these  cases  the  amino-acids  had  consistently 


50 


40 

Figure  18. 
The  solid  line  indicates  the  rate  of  re.spiration  at  25°  of  8  leaves  of  Helianthus  which 
were  kept  in  the  dark  for  43.5  hours  previous  to  putting  in  a  nitrogen-free  nutrient  solu- 
tion containing  7  per  cent  d-glucose,  as  per  table  49.  The"  broken  line  indicates  the  rate 
of  respiration  at  25°  of  8  similar  leaves  which  were  kept  in  the  dark  for  42.75  hours  pre- 
vious to  putting  in  a  nitrogen-free  nutrient  solution  containing  7  per  cent  glucose  and 
0.11  per  cent  glycocoll,  as  per  table  50.  The  ordinate  represents  mg.  CO2  per  hour  per 
gram  dry  material  and  the  absci.ssa  the  time  in  hours. 


70  STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 

increased.  In  table  27  and  figure  12  is  also  given  a  case  of  decreased 
respiration  rate  accompanied  by  increased  sugar-content  and  de- 
creased amino-acids.  However,  in  table  12  were  given  the  results 
of  the  carbohydrate  and  amino-acid  determinations  of  leaves  from 
plants  which  were  kept  in  the  dark,  showing  the  gradual  decrease 
in  carbohydrates  and  increase  in  amino-acids.  When  these  plants 
were  again  placed  in  the  light  the  reverse  process  occurred;  there 
was  an  increase  in  sugars  and  a  decrease  in  amino-acids. 


Figure  19. 
Rate  of  respiration  subsequent  to  periods  of  illumination.  Six  leaves  of  Helianthus 
at  25°;  petioles  in  a  nitrogen-free  nutrient  solution  containing  7  per  cent  d-glucose.  The 
solid  lines  indicate  the  CO2  emission  in  the  dark,  the  dotted  lines  indicate  the  periods 
during  which  the  leaves  are  illuminated.  The  ordinate  represents  mg.  CO2  per  hour 
per  gram  dry  material,  the  abscissa  the  time  in  hours. 

There  remains,  therefore,  to  show  the  effect  on  the  rate  of  respira- 
tion of  temporarily  decreasing  the  amino-acids  while  the  carbohy- 
drate-content remains  high.  It  has  been  shown  that  the  effect  of 
Ught  is  to  decrease  the  amino-acids.  The  following  experiment  was 
carried  out  so  that  the  leaves  which  from  the  beginning  of  the  experi- 
ment were  fed  d-glucose  were  illuminated  for  6  to  7  hours  after 
having  been  in  the  dark  for  about  25  hours.  Thus  during  the  course 
of  the  experiment  there  was  a  period  of  illumination  of  7  hours 
after  the  twenty-fifth  hour,  another  period  of  illumination  of  6  hours 
after  the  fiftieth  hour,  and  a  third  period  of  illumination  after  the 
seventy-fourth  hour.  As  a  source  of  illumination  there  was  used  a 
750-watt  tungsten  filament  lamp  at  40  cm.  distance.  The  glass 
cover  of  the  respiration  chamber  was  below  the  level  of  the  water 
in  the  thermostat.  The  results  of  this  experiment  are  given  in 
table  52  and  figure  19. 

During  the  periods  of  illumination  the  amount  of  carbon  dioxid 
absorbed  was  very  low.     This  would  indicate  that  a  large  part  of 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


71 


the  carbon  dioxid  of  respiration  was  fixed  through  photosynthesis. 
The  carbohydrate-content  of  the  leaves,  at  the  end  of  the  periods 
of  illumination,  was  therefore,  in  all  probability,  higher  than  at  the 
beginning  of  illumination.  It  must  also  be  borne  in  mind  that  the 
petioles  of  the  leaves  were  in  a  7  per  cent  d-glucose  solution  during 
the  entire  experiment. 

Table  52. — Rate  of  emission  of  CO2  by  6  leaves  of  Helianthus  annuus  at  25°;  petioles  in 
nitrogen-free  nutrient  solviion  containing  7  per  cent  d-glucose. 
The  leaves  weie  illuminated  during  periods  Nos.  5,   10,  and  15.     CO2  absorbed  in  Ba(0H)2 
solution  0.12170  normal,  125  c.  c.  of  which  has  the  equivalent  of  0.3346  gram  CO2. 


No. 

Time. 

Hrs. 

Total 
hours. 

Observed 
resist- 

Gram CO2 
equivalent 
of  125  c.  c. 

Gram 
CO2 

Mg. 
CO2 

Mg.  CO2 
per  hour 
per  gram 

ance  in 
ohms. 

Ba(0H)2 

absorbed. 

per 
hour. 

dry 

solution. 

weight. 

0 

2''30'"  p.m.  to  3''30°>  p.m. 

1 

1.0 

1 

3i'30'-  p.m.  to  9''30'"  p.m. 

6 

7.0 

71.3 

0 . 2436 

oiogio 

i5!l6 

7!38 

2 

Qi-SO^  p.m.  to  3''30"'  a.m. 

6 

13.0 

72.9 

.2376 

.0970 

16.16 

7.87 

3 

3^3(y  a.m.  to  9''30'"  a.m. 

6 

19.0 

73.2 

.2366 

.0980 

16.33 

7.95 

4 

g'^SO'^a.m.  to  3''30"'  p.m. 

6 

25.0 

70.7 

.2456 

.0890 

14.83 

7.22 

5 

3^30"' p.m.  to  10''30°'p.m. 

32.0 

60.4 

.2920 

.0426 

6.08 

2.96 

6 

10''30'"  p.m.  to  l^'SO'"  a.m. 

3 

35.0 

59.5 

.2978 

.0368 

12.26 

5.97 

7 

l^3(y^  a.m.  to  7i'30°'  a.m. 

6 

41.0 

67.9 

.2570 

.0776 

12.93 

6.29 

8 

7''30"»  a.m.  to  10^30"  a.m . 

3 

44.0 

60.0 

.2948 

.0398 

13.23 

6.44 

9 

lQl^3(y^  a.m.  to  4''30°'  p.m. 

6 

50.0 

69.1 

.2524 

.0822 

13.70 

6.67 

10 

4''30'°  p.m.  to  10''30"p.m. 

6 

56  0 

60.3 

.2930 

.0416 

6.93 

3.37 

11 

10''30"' p.m.  to  l»'30°'a.m. 

3 

59.0 

60.1 

.2938 

.0408 

13 .  60 

6.62 

12 

1''30'"  a.m.  to  7'»30"'  a.m. 

6 

65.0 

68.5 

.2550 

.0796 

13.26 

6.45 

13 

7'»30'"a.m.  to  10''30"a.m. 

3 

68.0 

60.4 

.2920 

.0426 

14.20 

6.91 

14 

10''30™a.m.  to  4'^30™p.m. 

6 

74.0 

70.5 

.2466 

.0880 

14.66 

7.14 

15 

4'>30^  p.m.  to  10''30™  p.m. 

6 

80.0 

59.5 

.2978 

.0368 

6.13 

2.98 

16 

10^30™  p.m.  to  li'30°'  a.m. 

3 

83.0 

59.8 

.2960 

.0386 

12.86 

6.26 

17 

l^SO"'  a.m.  to  7i»30°  a.m . 

6 

89.0 

68.0 

.2570 

.0776 

12.93 

6.29 

18 

7''30°'  a.m.  to  l''30°'  p.m. 

6 

95.0 

68.0 

.2570 

.0776 

12.93 

6.29 

The  experiment  shows  clearly  that  following  a  period  of  illumi- 
nation there  is  a  decrease  in  the  rate  of  respiration;  with  continued 
darkness  the  rate  rises  slowly  again  until  the  next  period  of  illumi- 
nation. It  must  be  remembered  that  under  the  conditions  of  the 
experiment  the  leaves  had  a  large  supply  of  available  sugar  in  the 
form  of  d-glucose.  From  the  experiments  which  have  been  described 
it  becomes  evident  that  there  are  at  least  two  factors  affecting  the 
rate  of  the  respiratory  process.  One  of  these  is  the  supply  of 
carbohydrates  and  the  other  is  the  amount  of  amino-acids.  A  change 
in  either  of  these  factors  alters  the  rate  of  respiration.  Under  most 
circumstances  light  seems  to  affect  these  two  factors  in  an  opposite 
manner:  the  carbohydrates  are  increased  by  the  photosynthetic 
activity  of  light,  while  under  these  conditions  the  amino-acids 
decrease.  It  is  conceivable  that  in  this  manner  the  rate  of  respiration 
tends  to  be  equalized  under  varying  external  conditions. 


72  STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 

The  increased  rate  of  growth  in  the  dark,  which  has  been  very 
commonly  observed,  can  also  in  a  measure  be  explained  on  the  basis 
of  the  foregoing  experiments.  In  the  dark  there  is  an  accumulation 
of  amino-acids  in  most  plant  parts.  Given  an  adequate  suppl}'^  of 
carbohydrates,  stored  in  the  stems  and  other  parts  of  the  plant, 
an  increase  in  amino-acids  resulting  from  the  absence  of  light  would 
cause  an  increased  respiratory  rate.  The  behavior  of  an  entire 
plant  has  been  described  in  the  first  experiment,  under  table  8  and 
figure  7.  It  is  therefore  suggested  that  the  augmented  respiratory 
activity  is  at  least  one  important  factor  contributing  to  the  increased 
rate  of  growth  during  periods  of  darkness. 

There  are  many  points  regarding  the  relation  of  carbohydrates 
and  amino-acids  to  light  which  require  further  study.  These  ques- 
tions seem  to  us  to  be  of  great  importance  to  the  problems  of  photo- 
synthesis and  are  now  being  subjected  to  investigation. 

It  would,  of  course,  have  been  highly  desirable  if  the  proteins  as 
well  as  the  amino-acids  could  have  been  determined  in  the  leaf 
material.  This,  however,  was  impossible,  largely  because  of  the 
relatively  small  amount  of  material  remaining  from  the  respiration 
determinations  and  from  the  analyses  for  amino-acids  and  sugars. 
In  view  of  the  fact  that  the  leaves  were  kept  in  a  nutrient  solution 
containing  no  nitrogen,  it  seems  highly  probable  that  the  amino- 
acid  increase  came  from  the  breakdown  of  the  proteins.  That  the 
leaves  contained  ample  proteinaceous  material  to  supply  the  increase 
in  amino-acids  becomes  evident  from  the  following  analyses.  A 
typical  dry-leaf  material  of  Heliarithus  contained  5.50  per  cent  of 
nitrogen  as  NH2,  determined  by  the  Folin  micro-Kjeldahl  method. 
The  water  extract  yielded  0.119  per  cent  nitrogen  as  NH2  by  the 
Van  Slyke  method,  representing  free  amino-acids,  and  0.463  per 
cent  nitrogen  as  NH2  by  the  Folin  micro-Kjeldahl  method.  Evi- 
dently some  nitrogenous  material  other  than  amino-acids  is  extracted 
by  means  of  water.  This,  however,  does  not  affect  the  Van  Slyke 
results.  Some  idea  of  the  amount  of  protein  in  the  dry-leaf 
material  can  be  gained  from  the  results  of  the  hydrolysis  with  20 
per  cent  hydrochloric  acid.  This  hydrolyzed  material  contains  total 
nitrogen  as  NH2  of  5.03  per  cent  by  the  FoUn  micro-Kjeldahl  method. 
The  discrepancy  between  this  figure  and  the  5.50  per  cent  represent- 
ing the  total  nitrogen  of  the  material  which  had  not  been  previously 
treated  with  20  per  cent  acid  must  represent  the  humin  formation 
resulting  from  the  reaction  of  the  amino-acids  with  the  carbohydrate. 
The  hydrolyzed  material  yielded  amino  nitrogen  by  the  Van  Slyke 
method  of  2.88  per  cent.  Although  the  latter  value  is  undoubtedly 
a  little  low  on  account  of  the  humin  formation  during  hydrolysis, 
it  represents  a  considerable  reserve  of  protein  when  compared  with 
the  original  amino  nitrogen  in  the  water  extract  of  0.119  per  cent. 
Thus  the  hydrolyzed  material  contains  about  twenty  times  more 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS.  73 

amino-acids  than  the  water  extract.  This  gives  an  indication  of  the 
amount  of  protein  in  the  leaf  as  compared  with  the  amino-acids.  A 
considerable  number  of  analyses  such  as  the  one  just  described  were 
carried  out,  all  of  which  represented  about  the  same  proportions,  but 
as  the  results  have  not  a  real  quantitative  value  they  are  omitted  here. 

Special  tests  were  also  made  to  determine  whether  there  was  an 
accumulation  of  nitrates  in  the  leaves  which  had  been  kept  in  the 
dark.  However,  no  indication  could  be  found  that  this  was  the 
case.  In  fact,  no  tests  for  nitrates  could  be  obtained  in  the  water 
extracts.  Similarly,  the  amount  of  ammonia  was  so  small  that  it  can 
be  considered  as  insignificant.  The  ammonia  was  determined  by 
treating  the  leaf  powder,  suspended  in  water,  with  magnesium  oxide 
and  distilling  in  a  stream  of  air  at  35°  to  40°  under  reduced  pressure, 
15  mm.     The  distillate  was  taken  up  in  standard  acid  solution. 

It  is  well  known  that  with  an  ample  supply  of  carbohj^drates, 
protein  synthesis  can  take  place  in  the  leaf  in  the  dark  from  in- 
organic nitrates.^  The  observation  that  light  acts  favorably  on 
protein  synthesis — more  than  three  times  the  amount  which  is 
formed  in  the  dark — has  been  made  by  a  number  of  workers, ^ 
Since  it  had  been  shown  that  protein  synthesis  from  inorganic  nitrogen 
compounds  occurs  only  in  the  presence  of  an  abundance  of  carbo- 
hydrates, the  beneficial  influence  of  light  was  attributed  to  photo- 
synthetic  elaboration  of  carbohydrates  by  means  of  light.  That  this 
was  not  the  only  interpretation  to  be  given  to  experiments  showing 
the  favorable  action  of  light  on  protein  syntheses  was  made  evident 
by  the  experiments  of  Godlewski.^  He  showed  that  protein  synthesis 
from  nitrates  was  very  much  greater  in  the  light,  even  when  photo- 
synthesis was  largely  excluded  by  keeping  the  plants  in  a  carbon- 
dioxid-free  atm.osphere,  and  that  his  wheat  seedling  grown  under 
these  conditions  contained  not  only  as  much  protein  as  was  in  the 
original  seeds,  but  had  formed  a  certain  amount  over  and  above 
this.  Such  was  not  the  case  with  the  seedHngs  grown  in  the  dark. 
Evidently,  then,  light  exercises  a  very  direct  influence  on  protein 
synthesis.  These  observations  have  led  to  much  experimentation 
and  extensive  speculation  in  an  endeavor  to  ascertain  the  mode  of 
synthesis  of  the  complex  proteins. 

Emmerling^  and  others  consider  that  amino-acids  are  the  first 
products  of  nitrogen  assimilation  in  the  leaves.  However,  this  part 
of  the  problem  is  still  largely  in  the  speculative  stage  and  little 
direct  experimxcntal  evidence  is  available.  That  leaves  take  up 
amino-acids  and,  in  the  presence  of  sugar,  increase  in  protein- 
content  was  demonstrated  in  a  qualitative  manner  by  Hansteen.^ 

'  Zaleski.W.  Ber.  deutsch.  hot.  Ges.,  15, 536-542  (1897).  Prianischn'ikow,  D.  lbid.,17,151  (1899). 
-  Laurent,  E.,  and  M.  Mahchal.     Bull,  de  I'Acad.  Roy.  de  Belgique,  32,  55  (1903). 
» GoDLEWSKi,  E.     Bull.  Acad.  Science  de  Cracovie,  313  (1903). 

*  Emmerling,  E.     Landw.  Versuch.  Stat.,  34,  113  (1880). 

*  Hansteen,  B.     Jahrb.  wiss.  Bat,  33  417  (1899). 


74 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


From  the  experiments  of  Saposchnikowi  it  appears  that  excised 
leaves  rapidly  take  up  asparagine  and  in  the  light  show  an  increase 
in  protein-content. 

The  increase  of  amino-acids  in  leaves  kept  in  the  dark  offers  the 
key  to  the  interpretation  of  some  observations  of  long  standing 
which  have  never  been  adequatelj^  explained.  In  an  excellent  paper 
on  the  chemistry  and  physiology  of  foliage  leaves,  published  by 
Brown  and  Morris^  in  1893,  determinations  are  reported  of  the 
increase  of  diastatic  activity  of  leaves  kept  in  the  dark.  Thus,  for 
example,  in  experiments  with  Hydrocharis  morsusrance  they  found : 

Table  53. 


Diastatic 
activity. 

Increase  in 
diastase. 

After  full  insolation 

In  darkness  47  hours 

In  darkness  96  hours 

p.ct. 
0.267 
0.476 
0.676 

p.d. 

"78.2 
153.1 

The  explanation  given  for  this  phenomenon  by  Brown  and  Morris 
is,  in  brief,  that  the  protoplasm  elaborates  diastase  according  to 
the  requirements  of  the  leaf. 

"As  long  as  conditions  are  favorable  for  assimilation,  the  leaf-cells  are  supplied 
with  an  abundance  of  newly  assimilated  materials  and  so  plentifully  that  the  supply 
exceeds  their  powers  of  metabolism  and  translocation.  The  excess  of  nutritive 
material  is  in  part  at  least  deposited  as  starch.  At  this  period  there  is  little  or  no 
elaboration  of  diastase  of  the  protoplasm,  probably  none  at  all  in  those  cells  in  which 
starch  deposition  is  in  active  progress.  When  the  light  fails,  and  assimilation  falls 
off,  the  living  cells  speedily  use  up  or  translocate  the  excess  of  the  soluble  assimilative 
products,  e.  g.,  cane  sugar,  and  begin  to  draw  their  supplies  from  the  reserve  of  starch. 
To  enable  them  to  do  this  effectually,  the  somewhat  starved  protoplasm  now  com- 
mences to  elaborate  the  needed  diastase  more  rapidly,  and  this  secretion  becomes 
still  more  marked  as  the  starvation  point  of  the  cell  is  neared." 

It  seems  to  us  that  this  argument  ascribes  to  protoplasm  final 
causes  beyond  the  justification  of  the  experiments,  in  a  manner 
already  briefly  considered  in  the  introductory  discussion  of  this 
paper.  The  fact  that  amino-acids  increase  in  the  leaves  kept 
in  darkness  has  been  established  repeatedly.  The  accelerating 
influence  of  amino-acids  on  the  diastatic  activity  as  determined  by 
Sherman  and  others  offers  a  more  direct  explanation  of  the  periodic 
variations  of  diastase  in  leaves  than  the  one  originally  advanced 
by  Brown  and  Morris.  Thus  an  increase  in  diastatic  activity  of 
leaves  which  had  been  kept  in  the  dark  would  simply  mean  that  the 
amino-acids  in  the  leaves  had  increased  and  therebj^  produced 
conditions  which  are  favorable  to  diastatic  activity. 

'  Saposchnikow,  W.     Bot.  Zentrbl.,  63,  246  (1895). 

'  Bbown,  H.  T.,  and  G.  H.  Morris.     Jour.  Chem.  Soc.  London,  63,  644  (1893). 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS.  75 

8.   The  Action  of  Animo-Acids  on  Sugars. 

In  searching  for  a  possible  chemical  explanation  of  the  stimulating 
effect  of  amino-acids  on  the  respiration  of  leaves,  the  action  of  amino 
acids  on  sugars  deserves  some  consideration.  In  this  regard  the 
work  of  Maillard^  has  received  considerable  notice  as  offering  the 
key  to  the  amino-acid-carbohydrate  and  protein  relation  in  living 
organisms.  By  the  action  of  amino-acids  on  glj^cerine,  Maillard 
obtained  substances  to  which  he  ascribed  the  properties  of  poly- 
peptides. Subsequ:ently  he  studied  the  action  of  amino-acids 
on  a  variety  of  sugars  and  obtained  the  well-known  reactions  which 
lead  to  the  formation  of  humin  compounds.  It  is  very  questionable 
whether  these  reactions  can  find  application  to  living  organisms 
such  as  leaves.  Maillard  used  very  concentrated  solutions,  e.  g., 
4  grams  of  glucose  in  3  to  4  c.  c.  of  water  and  1  gram  of  glycocoll. 
Moreover,  the  reaction  proceeds  only  very  slowly  at  ordinary  tem- 
perature, so  that  his  conditions  can  not  be  considered  as  having 
direct  biological  significance. 

A  solution  of  d-glucose,  5.6853  grams,  in  100  c.  c.  water  in  a  2-dm. 
tube,  gave  a  rotation  a=  -H5.50  at  10°.  To  50  c.  c.  of  this  solution 
0.2401  gram  of  glycocoll  was  added;  the  rotation  of  this  solution  was 
a=  +5.47°.  After  24  hours  both  solutions  gave  the  same  rotation. 
The  d-glucose  solution  containing  the  glycocoll  was  heated  for  4 
hours  at  90°  with  a  reflux  condenser.  After  making  up  carefully  to 
volume,  the  rotation  was  practically  unchanged,  a  =+5.52°. 

The  experiment  was  repeated  with  d-levulose.  To  a  solution 
of  8.9675  grams  d-levulose  in  100  c.  c.  water  was  added  0.5002  gram 
glycocoll.  This  gave  a  rotation  in  a  2-dm.  tube  of  a  =—16.50°. 
After  heating  on  a  boiling  water-bath  for  5  hours  the  solution  was 
very  slightly  yellow  and  gave  a  corresponding  rotation  of  a  =  — 16.40°. 

It  is  therefore  very  doubtful  whether  the  amino-acids  in  this 
dilution  have  any  influence  toward  the  mutual  transformation  of  the 
hexoses  such  as  is  exerted  by  the  weak  alkalies.  Nor  does  there 
seem  to  be,  under  the  conditions  of  dilution  and  temperature  em- 
ployed in  these  experiments,  any  other  profound  action  on  the  sugars. 

The  foregoing  experiment  with  solutions  of  glycocoll  and  d-levulose 
were  repeated,  using  instead  of  water  the  nitrogen-free  mineral 
nutrient  solution  employed  in  the  respiration.  Also  under  these 
conditions  the  rotation  remained  unchanged  in  the  solution  kept  at 
ordinary  temperature  as  well  as  in  one  heated  on  the  boiling  water- 
bath  for  2  hours.  Similarly,  only  negative  results  were  obtained  in 
experiments  with  solutions  of  d-glucose  and  glycocoll  to  which  was 
added  the  juice  obtained  by  thoroughly  grinding  Helianthus  leaves 
with  quartz  sand. 

1  Maillard,  L.  C.     Ann.  de  Chimie.  (9  s^rie.),  5,  258-317  (1918);  2,  210-268  (1914).     Ivanoff, 
N.  N.     Biochem.  Zeitschr.,  120.  1-80  (1921). 


11.  THE  INTERNAL  FACTOR  IN  PHOTOSYNTHESIS. 
INTRODUCTORY  DISCUSSION. 

It  has  been  realized  for  some  time  that  in  the  photosynthetic 
process  taking  place  in  chlorophyllous  leaves  there  is  an  essential 
internal  factor  the  nature  of  which  has  thus  far  not  been  discovered. 
That  such  a  factor  exists  is  concluded  not  only  from  the  failure  of  all 
attempts  which  have  been  made  to  reproduce  photosynthesis  outside 
of  the  living  cell,  but  recently  also  from  direct  experiments  with  living 
leaves.  The  existence  of  such  a  factor  is  especially  noticeable  under 
circumstances  where  the  rate  of  the  photosynthetic  activity  varies 
quite  independently  of  external  conditions.  The  nature  of  this 
factor  and  the  seat  of  its  activity  have  been  given  a  large  variety 
of  purely  hypothetical  explanations. 

In  the  course  of  investigations  on  certain  phases  of  the  problem 
of  photosynthesis  which  have  been  in  progress  for  a  number  of  years, 
it  was  recognized  that  a  better  understanding  of  the  internal  factors 
affecting  respiration  was  a  prerequisite  to  a  rational  interpretation 
of  photosynthesis.  Considerable  information  on  the  nature  of  car- 
bohydrate consumption  in  leaves  has  been  gained  and  has  aided 
materially  in  the  experimental  work  on  photosynthesis.  It  now 
seems  highly  probable  that  a  solution  of  the  problem  of  the  internal 
factor  in  photosynthesis  can  be  found  in  the  intimate  interrelation 
between  photosynthesis  and  respiration. 

Pantaneli^  maintained  that  in  photosynthesis  the  major  role  is 
to  be  ascribed  to  the  protoplasmic  function  of  the  colorless  com- 
ponents of  the  chloroplasts.  Willstaetter  and  Stoll,^  in  their  splendid 
and  thorough  investigation  of  the  relation  of  the  chlorojihyll  com- 
ponents to  photosynthesis,  attempt  to  determine  whether  the 
differences  in  the  photosynthetic  activity  of  a  leaf  can  be  explained 
by  ascribing  a  dual  function  to  a  single  chemical  component  of  the 
chlorophyll.  That  this  factor  is  not  to  be  sought  in  the  chlorophyll 
components  or  in  such  physical  conditions  as  the  degree  of  dispersion 
of  the  chlorophyll  pigments  follows  from  the  great  and  irregular 
disproportionality  which  has  been  found  to  exist  between  the  chloro- 
phyll-content and  photosynthetic  activity.  Willstaetter  and  Stoll 
are  of  the  opinion  that  besides  the  chlorophyll  there  is  another 
chemical  agent  essential  to  the  photosynthetic. process.  The  func- 
tion of  this  agent  they  believe  can  be  ascribed  either  to  the  general 
behavior  of  the  plant  protoplasm  or,  in  attempting  to  enter  more 
deeply  into  its  chemical  nature,  it  must  be  ascribed  to  a  definite 

•  Pantaneli,  E.     Jahrh.  f.  wiss.  BoL,  39,  165  (1903). 

=  Willstaetter,  R.,  and  A.  Stoll.     Untersuchungen  uebor  die  Assimilation  der  Kohlensaeure, 
41-166  (1919). 
76 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS.  77 

component  of  the  protoplasm.  This  component  they  assume  to  be  a 
specific  enzyme  contained  in  the  stroma. 

So  far  as  these  conclusions  go  they  are  apparently  in  undeniable 
agreement  with  observational  facts,  for  it  is  well  known  that  even 
mild  disturbances  of  the  structure  of  the  chlorophyllous  cell  result 
in  inhibition  of  photosynthesis.  Of  great  interest  also  is  their 
observation  that  the  internal  factor  is  affected  by  temperature  in 
much  the  same  manner  as  most  enzyme  reactions. 

In  Willstaetter  and  Stoll's  experiments  on  the  effects  of  variations 
in  temperature  and  light  intensity  with  leaves  rich  and  poor  in 
chlorophyll,  the  disproportionahty  between  chlorophyll-content 
and  photosynthetic  activity  becomes  clearly  evident.  Moreover, 
[leaves  poor  in  chlorophyll  are  more  dependent  upon  variations  in 
light  intensity,  while  those  rich  in  chlorophyll  show  greater  variations 
with  temperature.  The  two  components  of  the  photosynthetic 
apparatus  thus  operate  in  such  a  manner  that  the  pigment  reacts  to 
variations  in  light  intensity  more  in  the  case  of  the  leaf  with  low 
chlorophyll-content  than  the  one  with  high  chlorophyll-content, 
while  the  internal  factor  reacts  more  directly  to  temperature  and 
manifests  itself  to  a  larger  degree  in  the  leaves  of  high  chlorophyll- 
content.  This  fact  can  be  expressed  in  terms  of  limiting  factors  in 
such  a  way  that  in  leaves  poor  in  chlorophyll  this  component  is  the 
limiting  factor,  while  in  leaves  rich  in  chlorophyll  the  internal  factor 
is  the  limiting  one,  and  complete  utilization  can  not  be  made  of  the 
higher  chlorophyll-content  on  account  of  the  relatively  low  activity 
of  the  internal  factor. 

The  most  valuable  experimental  data  on  this  subject  are  those 
pertaining  to  the  temperature  coefficients.  Unfortunately,  however, 
while  there  have  been  made  a  number  of  careful  determinations  of 
this  nature,  these  represent  a  variety  of  rather  isolated  observations 
on  different  plant  material,  with  varying  methods,  and  the  nutritional 
conditions  of  the  plant  have  not  been  taken  into  consideration  suffi- 
ciently. We  are  at  present  engaged  in  a  more  exhaustive  study  of 
the  subject  with  mature  leaves. 

Warburg^  found  that  the  temperature  coefficient  with  high  light 
intensity  and  high  carbon-dioxid  concentration  decreases  with  in- 
creasing temperature,  so  that  at  5°  it  is  4.3  and  at  32°  it  is  1.6.  With 
low  light  intensity  the  temperature  coefficient  is  about  unity.  These 
observations  can  be  explained  on  the  basis  of  Blackman's  theory  of 
limiting  factors;  but  on  careful  consideration  the  question  is  also 
raised  whether  this  theory  is  not  due  for  a  revision  or  extension,  not 
as  to  its  observational  basis,  but  rather  regarding  the  interpretation 
of  the  dynamics  of  the  factors  involved.     Temperature  relations 

1  Wahburg,  O.     Biochem.  Zeitschr.,  100,  230-370  (1919). 


78  STUDIES  IN  PLANT  RESPIKATION  AND  PHOTOSYNTHESIS. 

such  as  were  found  by  Warburg  are  not  at  all  uncommon  for  phys- 
iological processes.^  Becking^  has  recently  published  a  theoretical 
discussion  of  the  physical  factors  involved  in  the  temperature 
coefficients  of  vital  phenomena.  Osterhaut  and  Haas,^  on  the  basis 
of  determinations  of  the  temperature  coefficient  of  photosynthesis 
of  Ulva  rigida,  conclude  that  this  process  involves  two  reactions — 
one  a  light  reaction  with  a  low  temperature  coefficient  and  an  ordi- 
nary chemical  reaction  with  a  high  Qio. 

A  detailed  discussion  of  temperature  coefficients  can  not  be  entered 
upon  here.  Suffice  it  to  say,  however,  that  in  a  process  such  as 
photosynthesis  there  enters  such  a  large  number  of  factors  that 
the  question  naturally  arises  whether  the  very  small  number  of 
really  concordant  results  is  not  due  to  chance  or  more  probably  to 
the  choice  of  limited  conditions  to  the  exclusion  of  the  broader 
aspect  of  the  question.  The  many  factors  and  steps  which  con- 
tribute to  the  complete  process  naturally  produce  a  complicated 
situation,  and  determinations  of  temperature  coefficients  must 
represent  the  mean  of  a  number  of  reactions.  Thus,  while  the 
rate  of  photosynthesis  is  reduced  with  a  decrease  in  temperature, 
the  absorption  of  carbon  dioxid  at  25°  is  about  half  that  at  5°.* 

Recent  investigations  in  the  field  of  photochemistry  emphasize 
the  complex  nature  of  these  reactions,  and  it  will  require  an  enormous 
amount  of  experimental  data  before  the  kinetics  of  the  photosyn- 
thetic  process  are  made  clear.  Stokalasa's^  attempt  to  explain  the 
internal  factor  in  photosynthesis  on  the  basis  of  the  radioactivity 
of  potassium,  which  is  found  in  higher  concentration  near  the  chloro- 
plasts,  still  requires  much  experimental  evidence  before  it  is  beyond 
the  realm  of  the  purely  hypothetical. 

In  the  consideration  of  the  relation  between  respiration  and 
photosynthesis  it  is  primarily  the  mature  leaf  which  must  be  studied. 
The  principal  interest  in  photosynthesis  centers  about  the  leaf  which 
is  producing  carbohydrate  material  above  its  immediate  needs. 
For  this  reason  also  the  behavior  of  germinating  seeds  or  seedlings 
which  have  available  reserve  food  material  is  of  rather  secondary 
importance  in  the  study  of  photosynthesis. 

The  problem  of  photosynthesis  is  essentially  one  of  energy  transfer. 
Unfortunately,  however,  our  knowledge  of  the  energy  relations  in 
photosynthesis,  as  well  as  of  plant  respiration,  is  most  rudimentary. 
The  former  is  practically  confined  to  the  observations  of  Brown 

>  Fawcett,  H.  S.     University  of  California  Publications  in  Agricultural  Sciences,  4,  No.  8,  217 

(1921). 
2  Becking,  L.  B.     Dissertation,  University  of  Utrecht  (1921). 

sQsTEBHAUT,  W.  J.  V.,  and  A.  R.  C.  Haas.     Jour.  Gen.  Physiol,  1,  295-298  (1919). 
« WiLLSTAETTEK,  R.,  and  A.  Stoll.     I.  c,  p.  181. 
sStokalasa,  J.     Biochem.  Zeitschr.,  108,  159-184  (1920). 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS.  79 

and  Escombe^  and  of  Puriewitsch ;-  thus  far  no  measurements  have 
been  made  which  determine  directly  the  amount  of  radiant  energy 
used  in  photosynthesis.  Such  data  as  we  now  possess  have  been 
obtained  from  calculations  based  either  on  the  amount  of  carbon 
dioxid  absorbed  (Brown  and  Escombe)  or  on  the  heat  of  combustion 
of  the  photosynthate  (Puriewitsch).  Fundamentally  the  measure- 
ments of  Brown  and  Escombe  and  of  Puriewitsch  are  based  upon  the 
same  principle,  i.  e.,  taking  the  heat  of  combustion  of  the  photosyn- 
thate or  of  glucose,  or  its  equivalent  in  CO2,  as  a  measure  of  radiant 
energy  utilized.  The  notable  facts  in  Brown  and  Escombe's  studies 
are  that  only  a  small  proportion  of  the  energy  absorbed  by  the  leaf 
is  used  in  photosynthesis  and  that  the  amount  actually  used  is  a 
variable  quantity. 

Puriewitsch's  figures  exhibit  similar  variations  in  the  percentage 
of  radiant  energy  used  in  photosynthesis,  ranging  from  0.6  per  cent 
to  7.7  per  cent.  In  these  experiments  the  intensity  of  illumination 
was  high  and  normal  air  was  used,  so  that  the  carbon  dioxid  was 
probably  the  limiting  factor.  Under  such  conditions  it  is  to  be 
expected  that  the  percentage  of  radiant  energy  used  in  photosyn- 
thesis would  vary  inversely  with  the  intensity  of  illumination.  This, 
however,  is  not  the  case.  He  finds  that  the  longer  the  period  of 
illumination  the  lower  is  the  percentage  of  radiant  energy  utilized 
in  photosynthesis.  Thus  for  Polygonum  sacchalinense  the  time  and 
percentage  of  energy  used  are  as  follows:  1  hr.  20  min.  7.7  per  cent; 
2  hrs.  20  min.  3.7  per  cent;  5  hrs.  1.1  per  cent  and  2.5  per  cent. 
Since  the  ''time  factor"  in  photosynthesis  does  not  become  evident 
under  25°,  these  variations  can  probably  not  be  ascribed  to  it,  and 
we  must  assume  that  as  the  plant  accumulates  carbohydrates  less 
radiant  energy  is  utilized.  It  can  not  be  assumed,  as  Puriewitsch 
does,  that  in  his  experiments  this  decrease  is  due  to  the  accumulation 
of  the  photosynthate,  which  would  mean  the  interference  by  the 
well-known  Saposchnikoff  effect — in  brief,  an  inhibition  of  the 
photosynthetic  activity,  probablj'-  due  to  the  accumulation  of  car- 
bohydrates.3  This  effect  seems  to  become  apparent  in  land  plants 
only  after  more  prolonged  exposure  than  in  the  experiments  of 
Puriewitsch.  Unfortunately,  Puriewitsch  gives  no  data  as  to  the 
rate  of  respiration;  but  it  is  safe  to  assume,  from  the  experiments 
of  Matthaei''  and  our  own  on  the  relation  of  carbohydrate-content 

>  Brown,  H.  T.,  and  F.  Escombe.     Proc.  Roy.  Soc.  London,  B.  76,  29-111  (1905). 

2  Puriewitsch,  K.     Jahrb.  f.  wisa.  BoL,  53,  210-254  (1914). 

»  Saposchnikoff,  W.     Ber.  d.  deut.  bot.  Ges.,  11,  391-393  (1893). 

EwART,  A.  J.     Jour.  Linnean  Soc,  30,  439-443  (1896);  31,  573  (1897);  Ann.  of  Bot.,  11,  439- 
480  (1897). 
«Matthaei,  G.     Phil.  Trans.  Roy.  Soc.  London,  B.  197,  50  (1904). 

Borodin,  J.     Mem.  de  I'acad.  imp.  des  Sciences  de  St.  Petersburg  (Serie  7),  28,  4  (1881). 


80  STUDIES  IN  PLANT  BESPIRATION  AND  PHOTOSYNTHESIS. 

to  respiration,  that  as  photosynthesis  proceeds  the  rate  of  respiration 
increases.  It  would  appear,  then,  that  the  amount  of  radiant  energy 
utiHzed  in  the  photosynthetic  process  decreases  as  photosynthesis 
proceeds,  and  the  plant's  internal-energy  release  presumably  increases, 
due  to  higher  respiratory  activity  from  the  greater  available  car- 
bohydrate-supply. 

That  a  general  relationship  exists  in  leaves  between  the  rate  of 
carbohydrate  synthesis  and  consumption  has  been  recognized  for 
a  long  time.^     Boysen-Jensen^  summarizes  his  studies  as  follows: 

"In  Sinapis  the  intensity  of  the  CO2  assimilation  is  very  great,  rising  to  at  least 
6  mg.  CO2  per  50  cm.^  per  hour  at  20°.  Also  respiration  in  the  leaves  is  great,  about 
8  mg.  CO2  per  50  cm."  per  hour  at  20°.  The  point  of  equilibrium  between  CO2 
assimilation  and  respiration  lies  at  a  light  intensity  of  1.0  (Bunsen  units  XlOO). 
The  development  of  a  Sinapis  plant  is  very  weak.  In  44  weeks  the  dry-matter  con- 
tent rises  from  0.5  gram  to  38  grams  per  100  of  plant.  In  favorable  conditions  the 
daily  percentage  production  of  dry  matter  can  be  estimated  as  about  15. 

"In  Oxalis  the  maximal  intensity  of  CO2  assimilation  is  very  small,  about  0.8 
mg.  CO2  per  cm.^  per  hour  at  20°.  Also  respiration  of  the  leaves  is  very  small,  about 
0.1  to  0.2  mg.  CO2  per  50  cm.^  per  hour  at  20°.  The  point  of  equilibrium  between 
CO2  assimilation  and  respiration  lies  at  a  light  intensity  of  0.2.  The  daily  produc- 
tion of  dry  matter  is  at  2.1." 

If  an  actual  chemical  or  energetic  relationship  exists  between 
the  photosynthetic  and  respiratory  activities  of  the  leaf  it  should  be 
expected  that  any  disturbance  in  the  respiratory  activity  would  be 
reflected  in  photosynthesis.  The  observations  on  the  effect  of  re- 
duced oxygen  pressure  and  of  narcotics  on  photosynthesis  are  of 
special  interest  in  this  connection,  Boussingault^  and  Pringsheim* 
investigated  the  effect  of  lack  of  oxygen  on  the  rate  of  photosynthesis 
and  concluded  that  it  is  inhibited  thereby.  Willstaetter  and  Stoll,^ 
from  their  extensive  experiments  decide  that  oxygen  is  absolutely 
necessary  for  the  photosynthetic  reaction,  but  that  very  small 
amounts  of  oxygen  suffice.  This  small  amount  of  oxygen,  they 
claim,  need  not  be  present  as  free  oxygen,  but  as  loosely  bound, 
"dissoziabel  gebundener."  Although  their  experiments  were  appar- 
ently carried  out  with  great  care,  it  is  a  question  whether  they 
succeeded  in  removing  all  the  oxygen  which  is  occluded  in  the  leaves. 
This  fact,  however,  stands  out  clearly  in  their  experiments,  that  the 
more  perfectly  the  oxygen  has  been  removed  the  greater  is  the 
inhibition  of  the  photosynthetic  activity.  Also,  leaves  which  had 
been  previously  kept  in  the  dark  and  their  carbohydrate-content 
thus  reduced  showed  no  photosynthesis  in  an  atmosphere  freed  from 
oxygen.     From  these  experiments  it  appears  that  leaves  with  low 

1  EcKERSON,  S.     Bot.  Gaz.,  48,  224-228  (1909). 

2  Botsen-Jensen,  p.     Botanisk  Tidsskrift,  36,  21&-259  (1918). 
'  B0US8INGAULT,  J.  B.     Compt.  rend.,  61,  608  (1865). 

*  Pringsheim,  N.     Sitzber.  der  Preus.  Akad.  der  Wias.,  763  (1887). 

•  Willstaetter,  R.,  and  A.  Stoll.     I.  c,  344r-370. 


STUDIES  IN  PLANT -RESPIRATION  AND  PHOTOSYNTHESIS.  81 

carbohydrate-content,  i.  e.,  in  which  the  respiratory  energy  release 
is  very  low,  can  stand  the  absence  of  oxygen  less  easily  than  can 
leaves  with  higher  carbohydrate-content.  Also,  an  anaerobic  form 
of  respiration,  in  which  the  course  of  the  reaction  is  different  and  the 
energy  of  release  quite  low,  does  not  suffice. 

In  harmony  with  the  conception  of  the  dependence  of  photo- 
synthesis on  respiration  are  the  observations  of  the  effects  of  anes- 
thetics or  narcotics  on  photosynthesis.  Claude  Bernard  discovered 
the  inhibiting  effect  on  photosynthesis  of  chloroform,  and  this  fact 
has  been  repeatedly  confirmed.  ^  In  fact,  photosynthesis  is  far  more 
sensitive  to  the  action  of  chloroform  and  ether  than  is  respiration, 
so  that  amounts  of  these  anesthetics,  which  hardly  affect  the  rate 
of  CO2  emission,  exert  a  decidedly  inhibiting  effect  on  photosynthesis, 
and  with  higher  concentrations  the  capability  for  photosynthetic 
work  is  entirely  lost.  Willstaetter  and  Stoll-  have  shown  that  in 
leaves  which  have  lost  their  photosynthetic  power  the  four  chloro- 
phyll components  are  unchanged.  Unfortunately,  we  have  not  been 
able  to  gather  sufficient  data  on  the  nature  of  carbohydrate  metabo- 
lism in  leaves  during  narcosis  to  show  definitely  how  the  energy 
release  is  affected.  It  would  be  using  very  unreliable  evidence  to 
draw  conclusions  as  to  this  point  from  the  tropic  and  general  move- 
ment phenomena  or  other  responses  of  plants  under  the  influence  of 
narcotics. 

The  Very  interesting  experiments  of  Pelster'  bear  directly  on  the 
relation  of  photosynthesis  and  respiration.  He  found  that  while 
photosynthesis  is  much  lower  in  the  varieties  of  the  same  species 
containing  Uttle  chlorophyll,  there  is  no  direct  ratio  between  photo- 
synthetic activity  and  chlorophyll-content.  Furthermore,  the  fight- 
green  or  aurea  varieties,  with  low  chlorophyll-content,  also  have  a 
low  respiratory  activity  as  compared  with  the  normal  varieties. 
However,  here  also  there  is  no  direct  relation  between  respiration 
and  chlorophyll-content.  But  Pelster's  results  seem  to  show  a 
relation  between  respiratory  and  photosynthetic  activity.  The 
quotient  of  the  respiration  and  photosynthesis  values  of  the  light- 
green  types  are:  Ptelea  84.1/47.5  =  1.77,  Catalpa  58.8/34.2  =  1.72, 
Mirabilis  =  2.0,  Ulmus  =  2.0,  Populus  =  2.1,  while  Atiflex  showed 
the  very  low  quotient  1.3. 

The  findings  of  Pelster  can  be  explained,  of  course,  in  a  large 
measure  also,  on  the  ground  that  due  to  the  low  photosynthetic 

1  Bernard,  Cl.     Lecons  sur  les  phfenom^nea  de  la  vie,  278  (1878). 

Irving,  A.  A.     Ann.  of  Bot.,  25,  1077-1099  (1911). 

Kegel,  W.     Inaug.  Diss.,  Goettingen  (1905). 

EwART,  A.  J.     Jour.  Linnean  Soc,  31,  439  (1895). 

Bonnier,  G.,  and  L.  Magin.     Ann.  d.  Sci.  Nat.  Bot..  (7),  3,  14  (1886). 
s  Willstaetter,  R.,  and  A.  Stoll.     I.  c,  39. 
•  Pelster,  W.     Beitrdge  zur  Biologic  der  Pflamen,  11,  249-304  (1912). 


82  STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 

activity  the  leaves  have  a  relatively  low  supply  of  oxidizable  material, 
resulting  in  correspondingly  low  respiration  rates.  Therefore,  the 
experimental  conditions  in  the  investigations  of  Willstaetter  and 
StoU  were  destined  to  give  more  definite  results. 

In  just  what  manner  respiration  can  participate  in  the  photo- 
synthetic  process  it  is  as  yet  impossible  to  say  with  any  degree  of 
certainty.  That  the  heat  liberated  in  the  process  of  plant  respiration 
is  to  be  regarded  largely  as  an  energetic  waste  product  now  seems 
highly  probable. 

If  respiration  is  to  be  considered  as  a  producer  of  energy  for  the 
maintainance  of  the  so-called  life  processes,  the  potential  energy  of 
the  food  materials  can  not  be  converted  entirely  into  heat,  for  if 
that  were  the  case  the  heat  of  respiration  could  be  substituted  by  heat 
applied  from  without.  This,  of  course,  is  not  the  case.  There  must 
be  produced  in  the  course  of  respiration  other  forms  of  energy  which 
are  used  in  the  metabolic  processes  of  the  plant.  That  such  is  the 
case  was  indicated  in  the  old  experiments  of  Bonnier,^  who  found 
that  considerably  less  heat  was  evolved  than  could  be  expected  from 
the  respiratory  coefficient.  Further  calorimetric  studies  of  this 
kind  would  be  highly  desirable,  carried  out  on  the  basis  of  knowledge 
obtainable  regarding  the  transformation  of  the  carbohydrates  and 
other  food  material  and  with  the  application  of  modern  temperature- 
measuring  devices  of  high  accuracy.  While  such  calorimetric 
investigations  would  serve  as  excellent  guides  and  checks,  it  is 
evident  that  they  can  tell  us  little  of  the  more  intricate  details  of 
the  energy  release  in  respiration. 

Besides  heat,  then,  it  would  appear  that  the  plant  has  available 
and  uses  a  considerable  amount  of  energy  derived  from  the  breaking 
down  and  combustion  of  carbohydrates  and  other  food  material. 
Plant  physiologists  have  widely  accepted  the  dictum  that  this  energy 
is  converted  into  work  by  the  plant.  The  nature  of  the  work  which 
is  thus  performed  by  the  combustion  of  organic  substances  has  been 
described  in  only  vague  and  indefinite  terms,  although  it  is  the  basic 
problem  of  plant  life.  In  the  case  of  animals,  where  the  factors  of 
locomotion,  balance,  and  other  purely  mechanical  movements  are 
involved,  the  work  done  is  of  a  far  more  evident  nature.^  In  the 
plant,  however,  it  requires  rather  more  searching  perception  to 
determine  in  what  manner  this  release  of  energy  in  respiration  takes 
form,  for  it  is  a  strictly  chemical  phenomenon.  The  discussion  here 
is  limited  to  the  energy  liberated  by  the  breakdown  and  oxidation 
of  food  material.  The  tensions  and  pressures  related  to  turgor  or 
the  possible  mechanical  work  involved  in  the  activity  of  the  so-called 

1  BoNNiEE,  G.     Ann.  Sci.  Nat.  Bot.,  (7),  18,  1  (1893). 

Spoehr.  H.  A.     Carnegie  Inst.  Wash.  Pub.  No.  287,  21  (1919). 
«  Oppenheimek,  C.     Der  Mensch  als  Kraftmachine.    Die  Naturwiesenschaften,  23,  64-72  (1920). 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS.  83 

osmotic  energy  are,  of  course,  of  enormous  importance  in  the  life 
of  the  plant.  Similarly,  the  phenomena  dependent  upon  inhibition 
of  water  by  the  colloidal  material  and  surface-tension  energy  are 
of  great  significance  to  the  economy  of  the  plant  and  are  capable  of 
many  external  manifestations.  Nevertheless,  their  relation  to  the 
energy  transformations  of  food  material  can  be  considered  at  most 
to  be  only  an  indirect  one. 

In  general,  photosynthesis  and  respiration  bear  an  intimate 
association,  not  only  on  the  basis  of  direct  observation,  but  because 
oxidation  and  reduction  actions  in  the  living  organism  are  intimately 
connected  and  apparently  dependent  upon  the  same  or  very  closely 
allied  agents.  The  two  processes  of  photosynthesis  and  respiration, 
proceeding  in  opposite  directions,  may  be  related  either  on  the  basis 
that  the  energy  released  in  respiration  actually  aids  or  is  essential 
for  one  of  the  steps  of  the  reduction  process,  or  the  relation  may  be 
based  upon  the  action  of  an  enzyme  which  functions  in  both  reactions. 
Under  no  circumstances,  of  course,  can  all  the  energy  for  the  reduc- 
tion of  the  carbon-dioxid  come  from  the  oxidation  of  the  carbohy- 
drates.    An  extraneous  source  of  energy  is  essential. 

Thermodynamically,  the  contribution  which  respiration  could 
make  in  the  photosynthetic  process  would  naturally  be  relatively 
small,  so  that  this  amount  of  energy  could  at  best  serve  only  as  a 
partial  source. 

For  a  molecular  relationship  recourse  must  be  taken  to  our  modern 
conceptions  of  the  nature  of  carbohydrate  breakdown  or  glycosis. 
According  to  this  view,  as  has  been  elaborated  in  an  earher  publi- 
cation,^ precursory  to  oxidation  there  must  take  place  a  cleavage 
or  dissociation  of  the  molecule.  This  action  has  as  its  result  the 
formation  of  a  very  large  number  of  enormously  reactive  substances. 
These  pieces,  the  products  of  dissociation,  either  rearrange,  react 
with  each  other,  or  react  with  some  other  substance  present  in  the 
medium.  Now,  these  pieces,  on  account  of  their  enormous  reactivity, 
can  not  be  isolated.  It  is,  in  fact,  only  from  the  products  formed 
by  their  reaction  or  rearrangement  that  they  can  be  known.  They 
are,  nevertheless,  of  foremost  importance  in  the  chemical  reactions 
involved  in  carbohydrate  breakdown.  A  molecular  relation  of 
photosynthesis  and  respiration  would  depend  upon  the  activity 
of  these  intermediary  products  of  sugar  catabolism.  Just  as  these 
products  serve  as  the  building-blocks  from  which  other  compounds 
of  higher  potential  energy  may  be  formed  in  the  cell,  it  is  conceiv- 
able that  they  also  may  react  with  carbon  dioxid  or  with  some  of 
the  primary  products  of  the  photochemical  breakdown  of  carbon 
dioxid.^ 

»  Spoehr,  H.  A.     Carnegie  Inst.  Wash.  Pub.  No.  287,  5-24  (1919). 
"Siegfried,  M.,  and  S.  Howwjanz.     Zeit.  f,  Physiol.  Chem,  59,  376  (1909). 


84  STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 

One  of  the  most  remarkable  and  puzzling  features  of  the  carbohy- 
drate synthesis  in  the  plant  is  the  fact  that  the  sugars  found  in  nature 
are  confined  to  a  small  number  of  those  which  are  theoretically 
possible  on  the  basis  of  stereoisomeric  constitution.  A  conception 
of  the  manner  in  which  the  plant-cell  is  able  to  synthesize  carbohy- 
drates from  compounds  which  are  already  extant  in  the  cell  has  been 
suggested  by  E.  Fischer.  This  is  based  upon  the  fact  that  in  the 
artificial  synthesis  of  sugars  from  compounds  containing  a  smaller 
number  of  carbon  atoms,  by  means  of  the  cyanhydrine  reaction,  the 
influence  of  existing  stereoasymmetry  of  these  carbon  compounds  is 
exerted  on  the  final  product.  In  other  words,  an  asymmetric  carbon 
compound  yields  an  asymmetric  product.  In  the  same  way,  the 
reactive  products  of  the  primary  carbohydrate  dissociation  would 
also  be  asymmetric  substances,  and  these,  by  reaction  roughly  anal- 
ogous to  the  cyanhydrine  reaction,  have  the  power  of  uniting  with 
the  products  of  photochemical  decomposition  of  carbon  dioxid  and 
thereby  yield  an  asymmetric  product  in  the  form  of  d-glucose,  d-fruc- 
tose,  or  its  condensation  product,  sucrose.  As  these  substances  in 
turn  are  used  for  the  formation  of  the  large  variety  of  other  com- 
pounds found  in  plants,  they  would  serve  as  the  basis  of  the  asym- 
metry of  these. 

The  most  noteworthy  result  of  the  energy  release  of  respiration  in 
plants  is  the  formation  of  new  compounds.  These  compounds  may 
be  of  higher  energy  content,  although  they  may  not  again  serve  the 
plant  as  food  material,  but  enter  only  into  the  structural  or  plasmic 
elements  of  the  organism.  Or  respiratory  glycosis  may  serve  the 
plant  in  such  a  way  that  the  primary  products  thereof  are  substances 
on  to  which  carbon  dioxid  or  its  primary  splitting  product  can  be 
added  to  form  a  further  supply  of  hexose  sugars.  Given,  then,  a 
properly  functioning  respiratory  system,  together  with  the  necessary 
simpler  materials  and  an  apparatus  capable  of  utilizing  radiant 
energy,  the  plant  is  able  to  manufacture  not  only  the  large  variety 
of  substances  found  in  its  structural  elements,  but  also  to  add  organic 
material  derived  from  the  carbon  dioxid  of  the  air. 

METHODS  AND  APPARATUS. 
1.  The  Experimental  Material. 

The  experiments  on  the  photosynthetic  activity  were  carried  out 
with  single  excised  leaves.  For  this  work  leaves  of  the  Canada 
Wonder  bean  and  of  the  sunflower  were  used.  The  methods  of 
culture  were  the  same  as  those  employed  for  the  material  used  in 
the  respiration  experiments  and  the  same  precautions  were  observed 
in  regard  to  using  only  perfect  leaves  and  in  the  method  of  cutting 
and   handling   the   leaves.     These   always   remained   in   perfectly 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


85 


healthy  condition  during  the  course  of  the  experiment.  A  consider- 
able number  of  experiments  was  carried  out;  but  as  these  in  each 
case  yielded  consistent  results,  only  one,  representative  of  the  altered 
conditions,  is  described  here. 

2.  The  Apparatus. 

The  method  employed  for  determining  the  rate  of  photosynthetic 
activity  was  that  based  upon  the  differential  determination  of 
carbon  dioxid.  A  stream  of  air  containing  a  known  concentration 
of  carbon  dioxid  was  passed  over  the  leaf.  The  rate  of  carbon- 
dioxid  emission  from  the  leaf  when  this  was  in  the  dark  was  first 
determined;  this  gave  the  value  for  the  rate  of  respiratory  activity. 
The  leaf  was  then  illuminated  and  the  amount  of  carbon  dioxid  in 
the  gas-stream  after  it  had  passed  over  the  leaf  was  determined. 


Figure  20. 
Arrangement  of  apparatus  for  study  of  the  rates  of  photosynthesis.     The  entire  apparatus 
is  in  a  constant-temperature  room.     The  leaf-container  is  in  a  water-thermostat,  which  is  elec- 
trically heated,  controlled,  and  stirred. 

Thus  the  total  amount  of  carbon  dioxid  fixed  by  the  leaf  could  be 
calculated.  The  rate  of  the  gas-stream  was  maintained  absolutely 
constant  throughout  the  course  of  the  experiment.  The  entire 
apparatus  was  in  a  constant-temperature  room  which  was  ther- 
mostatically controlled.  The  leaf  was  in  a  container  which  was 
placed  in  an  electrically  controlled  water-thermostat,  usually  at  24° ; 
the  room  was  kept  at  20°. 

For  these  experiments  a  single  excised  sunflower  or  bean  leaf  was 
used.  The  same  advantages  and  disadvantages  of  working  with 
excised  leaves  for  determining  rates  of  respiration  which  have  already 


86 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


been  discussed  apply  to  the  work  on  photosynthesis.  With  a  single 
leaf  the  illumination  in  relation  to  the  surface  of  the  leaf  can  be  more 
accurately  controlled  than  when  there  is  a  larger  number  of  leaves. 
In  this  way  the  leaf  can  at  all  times  be  kept  at  right  angles  to  the 
source  of  hght,  and  the  shading  by  other  leaves,  or  changing  of  the 
angle  of  incidence  of  the  light,  can  be  absolutely  avoided. 

The  same  precautions  of  culture,  cutting,  and  handling  of  the 
leaves  which  were  observed  in  the  work  on  respiration,  already 
described,  apply  to  the  material  used  for  this  work.  The  experience 
and  knowledge  gained  from  a  study  of  the  rates  of  respiration  and 
the  changes  of  material  under  varying  conditions  were  naturally  of 
great  value  in  a  study  of  the  photosynthetic  activity. 


N- 


S 

Figure  21. 
Container  for  leaf  used  in  photosynthesis  experiments.     E  shows  the  elevation 
and  a  leaf  through  the  glass  plates  with  tube  A'^,  containing  the  nutrient  solution. 
<S  is  the  cross  section  of  the  container.     T  is  the  transverse  section  showing  the 
glass  plates  G  and  the  mercury  seal. 

A  diagram  of  the  apparatus  used  in  these  experiments  is  shown 
in  figure  20.  As  has  been  stated,  the  experiments  were  carried  out 
in  a  dark-room  which  was  thermostatically  controlled  and  kept 
at  20°.  The  air  was  taken  from  a  large  gasometer.  This  was  first 
partially  filled  with  carbon  dioxid  in  amounts  as  required.  The 
carbon  dioxid  was  prepared  from  white  marble  and  hydrochloric 
acid  and  was  washed  through  a  solution  of  sodium  carbonate  and 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS.  87 

one  of  potassium  permanganate.  The  gasometer  was  then  filled 
with  air  drawn  from  out  of  doors.  Special  experiments  were  carried 
out  to  insure  that  there  was  no  stratification  of  carbon  dioxid  in  the 
gasometer  and  that  this  gas  was  of  the  same  concentration  at  the 
beginning  and  end  of  the  experiment.  The  gasometer  was  sealed 
by  means  of  heavy  mineral  oil. 

By  means  of  glass  tubing,  the  gasometer  was  connected  to  a  spiral 
of  metal  tubing  which  stood  in  the  water  of  the  thermostat,  so  that 
the  air  attained  the  temperature  of  the  bath  before  entering  the 
leaf-container.  The  connections  made  by  means  of  heavy  wall 
rubber  tubing  were  wired  and  covered  with  several  coats  of  Bakelite 
paint. 

For  these  experiments  a  single  leaf  was  placed  in  a  chamber  or 
cell  of  special  construction.  This  consisted,  essentially,  of  a  metal 
frame,  15  by  25  cm.,  two  sides  of  which  were  glass  plates,  5  mm. 
apart.  This  leaf-cell  is  shown  in  figure  21,  in  elevation  (E)  in  section 
(S)  and  in  transverse  section  ( T) .  The  upper  part  of  the  metal  frame 
carries  a  metal  trough,  similar  to  that  of  the  respiration  chamber, 
already  described.  This  trough  was  also  electroplated  with  copper 
and  nickel  and  covered  with  lacquer,  so  that  it  could  hold  mercury. 
Into  this  trough  fits  a  metal  cover  (C  in  fig.  21).  Distilled  water 
was  poured  over  the  surface  of  the  mercury,  and  when  the  cover  was 
placed  in  the  trough  a  completely  air-tight  seal  was  made.  Through 
the  cover  passes  a  metal  tube  by  means  of  which  the  air-stream 
enters  into  the  leaf-container.  A  narrow-bore  tube  through  the 
lower  part  of  one  edge  of  the  container  provides  the  means  for 
carrying  off  the  air-stream.  At  the  bottom  of  the  leaf-container  is  a 
glass  tube  with  a  flared  opening.  Into  this  was  placed  the  petiole 
of  the  leaf,  which  was  suppUed  with  nutrient  solution  through  the 
tube  N.  This  tube  was  filled  with  nutrient  solution  and  sealed  at 
the  upper  end  with  a  piece  of  rubber  tubing  and  a  screw-clamp. 
During  the  course  of  an  experiment,  the  level  of  the  solution  in  which 
the  petiole  stood  went  down;  this  was  adjusted  from  time  to  time 
by  opening  the  screw-clamp  at  the  upper  end  of  the  tube. 

To  the  exit-tube  of  the  leaf-container  is  attached  a  water-trap  con- 
taining phosphorus  pentoxide.  The  experiments  were  carried  out 
in  a  saturated  atmosphere.  This  was  assured  by  filling  the  gasometer 
with  moist  air;  moreover,  the  water  over  the  mercury  seal  and  the 
nutrition  solution  provided  ample  water-vapor.  The  tubes  through 
which  the  air-stream  passes  after  leaving  the  leaf-container  are  all  of 
small  bore,  2  mm.  It  was  therefore  essential  to  avoid  the  formation 
of  water-drops  by  condensation  in  these  tubes.  This  was  accom- 
plished by  means  of  the  water-trap  filled  with  phosphorus  pentoxide. 

From  the  leaf-container  the  air-stream  passed  to  the  control- 
valve.     This  is  shown  in  figure  22,  and  consists  of  a  very  fine  needle- 


88 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


valve  which  pierces  just  through  a  piece  of  lead  L.  By  means  of 
this  valve  the  rate  at  which  the  air-stream  passes  through  the  appa- 
ratus could  be  very  accurately  regulated.  The  valve  is  attached  to 
a  small  vessel  containing  a  little  phosphorus  pentoxide. 

From  the  control-valve  the  air-stream  passes  through  narrow-bore 
glass  tubes  to  the  distributing  tube.  This  consists  of  glass  stopcocks 
by  means  of  which  the  air-stream  can  be  directed  to  the  absorption 
tubes.  Each  stopcock  is  connected  to  a  narrow  glass  tube  slightly 
bent  up  at  the  end.  Over  these  tubes  are  fitted  the  absorption  tubes 
by  means  of  a  small  rubber  stopper.  The  air-stream  can  thus  be 
directed  from  one  absorption  tube  to  another 
without  interrupting  the  rate  of  flow. 

The  absorption  tubes  consist  of  10-bulb  glass 
tubes,  the  lower  end  of  which  is  slightly  bent. 
They  are  filled  by  means  of  a  pipette,  carefully 
graduated.  For  these  experiments  68.12  c.  c. 
of  barium  hydroxide  was  used  for  each  determina- 
tion. It  was  found  that  the  bulb-tubes  break  up 
the  air-bubbles  more  thoroughly  and  thus  assure 
more  complete  absorption  than  the  straight-walled 
Pettenkoffer  tubes. 

Fig.  22. — Needle  control  valve  by  means  of  which  the  rate  of  the 
au--stream  passing  through  the  apparatus  is  regulated.  The  valve  is 
shown  in  section;  a  fine  needle  pierces  the  sheet  of  soft  lead  L,  and 
is  raised  and  lowered  by  means  of  a  packed  screw. 


From  the  absorption  tubes  connection  is  made  to  a  bottle  which 
served  to  determine  the  rate  of  the  gas-stream.  This  bottle  is 
partly  filled  with  water,  over  which  is  a  layer  of  mineral  oil.  The 
glass  tube  from  the  absorption  tubes  extend  to  the  bottom  of  this 
bottle.  The  rate  of  the  gas-stream  is  determined  by  counting 
the  gas-bubbles  passing  through  the  water  in  the  bottle  during  a 
certain  period.  The  time  measurements  were  made  with  a  stop- 
watch, and  by  means  of  the  control-valve  the  rate  of  flow  could  be 
very  accurately  adjusted.  A  rate  of  70  bubbles  per  minute  repre- 
sented a  flow  of  310  c.  c.  per  hour.  Care  was  taken  to  maintain 
constant  the  pressure  in  the  aspirator.  This  was  indicated  by  a 
manometer  filled  with  aniline  oil.  Blank  experiments  showed  that 
with  the  constant  temperature  of  the  room  and  in  the  water  ther- 
mostat a  very  regular  flow  of  air,  and  consequently  of  carbon  dioxid, 
is  obtainable  and  obviates  the  use  of  cumbersome  and  inaccurate 
gasometers. 

The  aspirator  consists  of  an  inverted  galvanized-iron  cylinder 
which  is  drawn  out  of  a  vessel  of  water.  The  cyhnder  is  attached 
to  a  weight  by  means  of  a  light  wire  cable.     This  passes  over  the  rim 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


89 


of  a  bicycle  wheel  which  is  suspended  from  the  ceiUng  of  the  room. 
The  wheel  has  excellent  ball-bearings  and  is  fastened,  so  that  there 
is  no  play  and  very  little  friction.  As  the  weight  descends  it 
lifts  the  cylinder  out  of  the  water,  which  causes  the  air-stream  to 
be  drawn  through  the  entire  apparatus.  It  is,  however,  necessary 
to  make  an  adjustment  for  the  increasing  weight  of  the  cylinder,  as  a 
larger  portion  of  this  comes  out  of  the  water.  This  is  attained  by 
increasing  the  pull  on  the  other  end  of  the  cable  through  a  supple- 
mentary weight  placed  on  the  wheel.  When 
the  cylinder  is  completely  down  this  weight  is 
at  the  top  of  the  wheel,  exerting  no  pull.  As 
the  cylinder  is  drawn  out  of  the  water,  the 
wheel  turns  and  the  pull  of  the  supplemen- 
tary weight  increases  as  the  horizontal  dis- 
tance between  it  and  the  center  of  the  wheel 
increases.  Thus  the  increasing  weight  of  the 
cylinder  is  adjusted;  and  while  the  two  phe- 
nomena do  not  mathematically  balance  each 
other,  the  adjustment  can  be  made  sufficiently 
accurate  so  that  no  differences  in  the  rate  of 
the  air-stream  are  noticeable. 

The  length  of  time  for  each  observation 
was  usually  two  hours.  The  amount  of  car- 
bon dioxid  fixed  by  a  single  leaf  in  this  length 
of  time  is  very  small.  It  was  therefore  essen-  , 
tial  that  the  accuracy  of  the  method  of  de-  ^ 
termining  carbon  dioxid  be  correspondingly 
high.  For  these  experiments  the  same  princi- 
ple of  carbon-dioxid  determination  was  em- 
ployed as  in  the  respiration  experiments.  On 
account  of  the  shorter  periods  and  smaller 
amounts  of  carbon  dioxid  to  be  absorbed,  a 
smaller  range  in  the  specific  resistance  curve 
of  barium-hydroxide  solution  was  used. 
Greater  accuracy  was  attained  by  using  an 
electrolytic  cell  of  higher  resistance.  The 
original  barium-hydroxide  solution  was  0.1001 
normal,  and  in  the  absorption  of  the  carbon 
dioxid  this  was  reduced  to  about  0.086  nor- 
mal. For  each  determination  68.12  c.  c.  of 
solution  was  introduced  by  means  of  a  pipette 

Fig.  23. — Electrolytic  cell  for  determining  the  conductivity 
of  the  barium  hj'dioxide  solutions  used  to  absorb  the  CO2  in  the 
photosynthesis  experiments.  The  solution  is  protected  from  the 
air  by  a  soda-lime  tube. 


90  STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 

into  the  absorbing  tube,  and  the  method  of  calculating  the  carbon 
dioxid  for  each  period  was  essentially  the  same  as  in  the  respira- 
tion experiments.  A  curve  of  the  specific  resistance  for  concentra- 
tions of  0.1001  to  0.0860  normal,  similar  to  the  one  used  for  the 
respiration  work,  was  determined  by  means  of  the  high-resistance 
cell,  and  was  used  for  establishing  the  amount  of  carbon  dioxid 
absorbed. 

The  barium-hydroxide  solution,  after  absorption  of  the  carbon 
dioxid,  was  transferred  rapidly  to  narrow  bottles;  these  were  then 
stoppered  and  sealed  with  paraffine.  After  all  the  barium  carbonate 
had  settled  out,  the  conductivity  of  the  clear  supernatant  liquid 
was  determined  bj^  means  of  the  electrolytic  cell  shown  in  figure  23. 
This  is  essentially  a  pipette  form  of  cell  with  a  glass  stopcock,  and 
the  solution  is  protected  from  the  air  by  means  of  a  soda-Hme  tube. 
The  cell  had  a  resistance  of  1,950  ohms  when  filled  with  0.1  normal 
potassium-chloride  solution.  For  making  the  conductivity  deter- 
minations, the  cell  was  submerged  slightly  above  the  upper  electrode 
in  a  water  thermostat  kept  at  25°.  A  difference  of  1  ohm  in  observed 
resistance  represented  about  0.00014  gram  of  carbon  dioxid  when 
using  68.12  c.  c.  of  the  barium-hydroxide  solution.  For  the  deter- 
mination of  the  carbon  dioxid  in  the  different  periods  of  a  single 
experiment,  differences  of  at  least  several  ohms  were  observed, 
which  gives  an  indication  of  the  degree  of  accuracy  of  the  method. 

As  a  source  of  Ught  there  was  used  a  500  or  750  watt  tungsten 
Mazda  lamp.  The  distance  of  the  filament  from  the  leaf  was  35 
cm.,  and  the  light  traversed  about  8  cm.  of  water  in  the  thermostat. 
The  electric  lamp  was  so  placed  that  the  light  fell  on  the  leaf  at  right 
angles.  Immediately  above  and  fitting  over  the  top  of  the  glass 
bulb  of  the  lamp  was  a  metal  hood  in  the  form  of  an  inverted  funnel. 
This  hood  was  connected  by  means  of  a  5-cm.  pipe  to  an  electric 
suction  fan.  In  this  way  the  hot  air  surrounding  the  electric  lamp 
was  drawn  off. 

After  each  experiment  the  leaf  was  removed  from  the  leaf-container 
and  placed  in  a  photographic  printing-frame  with  blue-print  paper. 
From  the  print  thus  produced  the  area  of  the  leaf  was  determined 
by  means  of  a  planimeter. 

EXPERIMENTAL  RESULTS. 

The  problems  of  photosynthesis  must  be  met  in  succession.  It  is 
questionable  whether  the  ultimate  or  final  causes  can  be  discovered 
before  a  clearer  understanding  is  gained  of  the  workings  of  the 
immediate  causes  and  relationships.  The  introduction  by  Blackman 
of  the  conception  of  Hmiting  factors  into  the  experimentation  on  this 
phenomenon,  while  it  has  contributed  little  to  the  ultimate  causes 
involved,  has  aided  very  materially  in  the  experimental  investigation. 


STUDIf:S  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


91 


No. 


On  the  other  hand,  the  many  claims  of  simple  and  complete  explana- 
tion of  the  process,  based  usually  on  very  limited  and  inadequate 
experimentation,  have  done  more  to  befog  the  issues  than  to  clarify 
them.  The  factors  of  carbon-dioxid  supply,  light  intensity,  the 
quantity  of  chlorophyll,  temperature,  and  water-supply  have  all 
received  more  or  less  exhaustive  treatment.  Practically  all  of  these 
investigations  have  revealed  the  fact  that  there  are  other  internal 
factors  operative.  The  nature  of  these  internal  factors  is  unques- 
tionably complex,  though  there  is  no  reason  for  supposing  that  they 
are  not  amenable  to  physical  and  chemical  investigation. 

Table  54. — Rates  of  respiration  and  synthesis  of  an  excised  bean  leaf  {Canada  Wonder) 
kept  in  the  dark  64  hours  previous. 
In  atmospheric  air  at  the  constant  rate  of  the  air-stream  equal  to  0.64  mg.  CO2  per  2-hour 
period.     Observations  made  at  23.8°.     Area  of  leafj  =  111.84  sq.  cm.     Light  intensity  7,140  lux. 


Time. 


ai'lS" 
5'>30'' 

7h45m 

10  p.n 

12»'15" 
2''30" 

4h45m 

7  a.m 

9h20n) 

llMO" 
1''55'" 

4h05m 

6''20'° 
8''35'" 
10''45" 


Dec.  13: 

p.m.  to    5''15™ 

p.m.  to    7'^30™ 

p.m.  to    9H5"' 

a  to  12  n 

Dec.  14: 
"a.m.  to   2'' 1.5" 
a.m.  to    4'>30" 
a.m.  to    6''45" 

.  to  9  a.m 

a.m.  to  ll'^20'» 
°  a.m.  to  l'>40" 
p.m.  to  3*^55'" 
p.m.  to  6''05'" 
p.m.  to  8''20°> 
p.m.  to  10'»35™ 
'»p.m.tol2''45'» 


a.m. 
p.m 
p.m 
p.m 
p.m 
p.m 
a.m 


Dark 
Do. 
Do. 
Do. 

Do. 
Light 

Do. 

Do. 

Do. 
Dark 
Light 

Do. 

Do. 

Do. 
Dark 


Hrs. 


Total 
hours. 


66.00 
68.25 
70.50 
72.75 

75.00 
77.25 
79.50 
81.75 
84.08 
86.41 
88.66 
90.82 
93.07 
95.32 
95.48 


Mg. 
CO2 
ab- 
sorbed. 


1.34 
1.22 
1.12 
0.98 

0.89 
0.27 
0.10 
0.04 
0.05 
1.01 
0.17 
0.10 
0.09 
0.23 
1.38 


Respira- 
tion, mg. 

COj 
emitted. 


0.70 
0.58 
0.48 
0.34 

0.25 


0.37 


Photo- 
;  synthesis, 
mg.  CO2 
fixed.  . 


0.62 
0.79 
0.85 
0.84 


0.84 
0.91 
0.92 
0.78 


Respira- 
tion per 
sq.  cm.  per 
hour  in 
mg.  CO2. 


0.0063 
0.0051 
0.0042 
0.0033 

0.0022 


0.0034 


0.0066 


Photo- 
synthesis 

per  sq.  cm. 

per  hour  in 
mg.  CO2. 


0054 
0072 
0076 
0076 


0076 
0080 
0082 
0072 


The  course  of  carbon-dioxid  emission  by  leaves  as  the  supply  of 
available  carbohydrates  diminishes  has  been  described  in  the  first 
section  of  this  publication.  The  decrease  in  the  rate  of  carbon- 
dioxid  emission  is  a  well-known  feature  of  these  conditions.  How- 
ever, the  course  of  the  photosynthetic  process  under  these  circum- 
stances has  received  little  attention. 

1.  In  table  53  the  results  are  given  of  the  rates  of  respiration 
and  subsequent  photosynthesis  of  a  bean  leaf  which  had  previously 
been  kept  in  the  dark  for  64  hours.  The  carbohydrate  supply  had 
thus  been  very  appreciably  diminished  and  the  decreasing  rate  of 
carbon-dioxid  emission  is  evident.  For  the  experiment  atmospheric 
air  was  employed  and  throughout  the  experiment  the  temperature 
of  the  thermostat  was  kept  at  23.8°. 


92 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


After  the  leaf  had  been  in  the  dark  for  75  hours,  the  rate  of  respi- 
ration was  0.0022  mg.  CO2  per  square  centimeter  per  hour.  During 
the  subsequent  2  hours  of  photosynthesis  the  leaf  fixed  0.0054  mg. 
CO2  per  square  centimeter  per  hour.  Thereafter  the  rate  of  photo- 
synthesis rose,  and  after  86.41  hours  the  rate  of  respiration  had  also 


il 


_r 


Figure  24. 
Rates  of  respiration  and  photosynthesis  of  an  excised  leaf  of  Canada 
Wonder  bean  kept  in  the  dark  64  hours  previously,  as  per  data  given  in  table  53. 
The  values  above  the  zero  line  indicate  rates  of  photosynthesis;  those  below, 
the  rates  of  respiration.  The  ordinates  represent  mg.  CO2  fixed  and  emitted 
per  sq.  cm.  per  hour  X 10;  the  numbers  of  each  square  correspond  to  the  periods 
as  given  in  table  54. 

risen  to  0.0034  mg.  CO2  per  square  centimeter  per  hour.  Apparently 
the  carbohydrate-supply  had  been  increased  through  the  photosyn- 
thetic  activity.  During  the  next  periods  of  exposure  to  light  the 
photosynthetic  rate  continued  to  rise,  and  the  final  period  of  respi- 
ration shows  a  considerably  higher  rate.  The  photosynthetic  rates 
are  in  each  case  calculated  by  subtraction  from  the  last  preceding 
respiration  period.  This  undoubtedly  accounts  for  the  relatively 
low  values  of  the  last  photosynthetic  period  (Nos.  9  and  14),  for 
the  leaf  was  at  this  time  emitting  carbon  dioxid  at  a  higher  rate 
than  during  the  last  respiration  period.  If  the  last  photosynthetic 
rates  were  calculated  from  the  first  succeeding  respiration  period, 
the  rate  of  the  former  would  result  considerably  higher.     This  fact 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


93 


stands  out  and  has  been  observed  repeatedly,  that  under  the  con- 
ditions of  the  experiment,  the  rate  of  photosynthesis  of  a  leaf,  the 
store  of  carbohydrates  of  which  has  been  greatly  reduced,  is  initially 
low  and  rises  with  continued  exposure  to  light.     Thereby  the  respi- 

Table  55. — Rates  of  respiration  and  photosynthesis  of  leaves  of  Helianthus  annuus  at  34°. 

Taken  after  plant  had  been  exposed  to  sunlight  and  after  increasing  periods  of  darkness. 
In  atmospheric  air,  light  intensity  7.140  lux. 

fFirst  leaf,  106.55  sq.  cm.] 


No. 

1 
2 
3 
4 
5 
6 
7 

Time. 

Leaf 
ex- 
posed 
to- 

Hrs. 

Total 
hours. 

Mg. 
CO2 
ab- 
sorbed. 

Respira- 
bion,  mg. 

CO2 
emitted. 

Photo- 
synthesis 
mg.  CO2 
fixed. 

Respira- 
tion per 
sq.  cm.  per 
hour  in 
mg.  CO2. 

Photo- 

synthesis 
per  sq.  cm. 
per  hour  in 

mg.  CO2. 

March  5. 
6''15'"  p.m.  to    7''15'"p.m. 
7''27"  p.m.  to   9''27°'  p.m. 
qHO™  p.m.  to  ll''40°' p.m. 
11''55"'  p.m.  to  li'SS^a.m. 
2''05'»  a.m.  to    4''05"  a.m . 
4''15°' a.m.  to    6''15'"a.m. 
6''30"  a.m.  to    S^'SO™  a.m . 

Dark 

Do. 

Do. 
Light 

Do. 
Dark 

Do. 

2 
2 
2 
2 
2 
2 
2 

2.00 
4.20 
6.40 
8.65 
11.15 
13.31 
15.56 

1.82 
1.41 
1.39 
0.26 
0.24 
1.27 
1.22 

1.180 
0.770 
0.750 

0.63 
0.58 

1.130 
1.150 

0.0111 
0.0073 
0.0071 

0.0107 
0.0109 

0.0059 
0.0054 

" 

[Second  leaf,  93.97  sq.  cm.] 

8 
9 
10 
11 
12 
13 

March  6. 
7''30°'  p.m.  to  9  ''30°'  p.m. 
9''45'"  p.m.  to  ll''45°'  p.m. 
ll''55°  p.m.  to  1''55'"  a.m. 
2''05"a.m.  to    4''05°'a.m. 
4^15"  a.m.  to    6''15°'a.m. 
6''25'"  a.m.  to    8''25°'  a.m . 

Dark 

Do. 

Light 

Do. 
Dark 

Do. 

2 

2 
2 
2 

2 
2 

28.56 
30.81 
32.97 
35.13 
37.29 
39.45 

1.02 
1.03 
0.27 
0.23 
0.92 
0.90 

0.38 
0.39 

"o!28'" 
0.26 

"o!76 
0.80 

0.0040 
0.0041 

0.0080 
0.0085 

0.0029 
0.0028 

[Third  leaf.  98.29  sq.  cm.] 

14 
15 
16 
17 

March  10. 
4''05°'  p.m.  to    6''05'^  p.m . 
6''15°' p.m.  to    8''15'»p.m. 
8''25°'  p.m.  to  10''25°'  p.m . 
10''35'"  p.m.  to  12''35°'  a.m . 

Dark 
Do. 

Light 
Do. 

2 
2 
2 

2 

119.11 
121.27 
123.43 
125.59 

1.07 
1.06 
0.35 
0.43 

0.43 
0.43 

"o!7l' 
0.63 

0.0043 
0.0042 

0.0071 
0.0064 

[Fourth  leaf,  91.91  sq.  cm.] 

18 
19 
20 
21 

March  12. 
8''10'"  a.m.  to  10''10°'  a.m . 
10''25'"  a.m.  to  12''25'"  p.m . 
12»'35°'p.m.to2''35°'p.m. 
2h45m  pua.  to    4''45°'  p.m . 

Dark 
Light 
Do. 
Dark 

2 

2 
2 

2 

179.17 
181.42 
183.58 
185.74 

0.82 
0.52 
0.79 
1.77 

0.18 

"i^is" 

"0.30 
0.03 

0.0019 

0.0032 
0.0003 

0.0159 

ration  rates  also  show  an  increased  rate  and  subsequent  periods  of 
photosynthesis  exhibit  correspondingly  higher  rates.  The  results 
of  this  experiment  are  shown  graphically  in  figure  24. 

2    The  following  experiment  was  carried  out  in  a  different  manner. 
A  mature  leaf  was  cut  from  a  large,  healthy  sunflower  plant  which 


94 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


had  been  exposed  to  bright  sunHght  in  the  greenhouse  during  the 
two  previous  days.  The  leaf  was  taken  in  the  afternoon  and  placed 
immediately  in  the  apparatus  for  determination  of  the  respiration 
and  photosynthesis  rates.  The  entire  plant  was  covered  with  a 
large  hood  of  black  paper,  so  as  to  exclude  all  light  without  altering 
other  conditions.     On  succeeding  days  leaves  were  cut  from  this 


4)S_ 


ffi 


u 


Figure  25. 
Rates  of  respiration  and  photosynthesis  of  leaves  of  Helianfhus  annuua  at  24°,  after  increas- 
ing periods  of  darkness.     The  values  above  the  zero  line  indicate  rates  of  photosynthesis,  those 
below,  rates  of  respiration,  expressed  on  the  ordinates  in  mg.  CO2  fixed,  or  emitted,  per  sq.  cm. 
per  hour  X 10.     Data  taken  from  table  65. 


plant  and  their  respiration  and  photosynthesis  rates  determined. 
In  this  manner  leaves  were  used  which  had  been  attached  to  the 
plant  during  the  period  of  starvation  and  their  store  of  carbohydrates 
was  not  depleted  as  rapidly  as  when  they  were  cut  from  the  plant. 
Moreover,  the  temperature  of  the  greenhouse  in  which  the  covered 
plant  stood  did  not  rise  above  22°,  while  the  determinations  were 
carried  out  at  24°.     For  these  reasons  some  of  the  leaves  at  first 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


95 


show  a  slightly  higher  initial  rate  of  respiration  than  the  ones  which 
had  been  cut  and  placed  in  the  apparatus  in  the  preceding  period. 
Thus  the  first  leaf  was  taken  after  an  entire  day  of  insolation,  and 
the  succeeding  ones  after  26.56,  117.11,  and  177.17  hours  of  darkness. 
The  results  are  given  in  table  55. 

From  the  graph  (fig.  25)  in  which  these  determinations  are  plotted, 
it  is  evident  that  the  leaves  which  had  been  kept  in  the  dark  show  a 
continual  decrease  in  respiratory  activity.  Moreover,  the  amount  of 
carbon  dioxid  fixed  also  shows  a  decline  with  decreasing  respiration. 
In  the  last  two  periods  of  the  fourth  leaf  there  are  signs  of  internal 

Table  56.— Rates  of  respiration  and  photosynthesis  on  three  successive  days  of  an 
excised  leaf  of  Helianthus  annuus  kept  in  the  dark. 


CO2  concentration  was  nine  times  that  of  atmospheric  air;  temperature  24°;  light  7,140 
lux;  area  of  leaf,  86.43  sq.  cm.    The  air-stream  equals  5.30  mg.  CO2  per  hour. 

No. 

Leaf 
ex- 
posed 
to- 

Time 

Hrs. 

Total 
hours. 

Mg. 
CO2 
ab- 
sorbed. 

Respira- 
tion, mg. 

CO2 
emitted 
per  hour. 

Photo- 
synthesis, 
mg.  CO2 
fixed  per 
hour. 

Respira- 
tion per 
sq.  cm.  per 
hour  in 
mg.  CO2. 

Photo- 
synthesis 

per  sq.  cm. 

per  hour  in 
mg.  CO2 

1 
2 
3 

4 
5 
6 

7 
8 
9 

Dark 
Light 
Dark 

Dark 
T,ight 
Dark 

Dark 
Light 
Dark 

March  17. 
1H5"  p.m.  to  3''45"  p.m. . 
3''50™  p.m.  to  5''50"  p.m. . 
5''55™  p.m.  to  7*'55™  p.m. . 

March  18. 
gJ^OS-^a.m.  toll''05"a.m. 
ll*»10^a.m.  to  l^'lO^p.m. 
l''15'"p.m.  to3''15'"p.m.. 

March  19. 
3''15°  p.m.  to  Sills'"  p.m. . 
5^2(y^  p.m.  to  7»'20°"  p.m. . 
7''25°'  p.m.  to  9^25-"  p.m. . 

2 
2 
2 

2 
2 
2 

2 
2 
2 

2.000 
4.082 
6.166 

21.533 
23.616 
25.700 

49.70C 
51.783 
53.866 

20.76 
11.16 
20.61 

19.18 
13.31 
20.21 

16.61 
15.86 
16.26 

5.08 

0.0587 

4.80 

0.0554 

5.00 
4.60 

0.0578 
0.0531 

3.25 

0.0376 

4.81 
3.01 

0.0556 
0.0348 

0.38 

0.0044 

2.83 

0.0327 

disturbance.  The  leaf  had  a  sUghtly  mottled  appearance  and  was 
curled  in  on  the  edges,  so  that  the  very  high  rate  of  carbon-dioxid 
emission  in  the  final  period,  No.  21,  was  probably  due  to  protoplasmic 
disturbances.  In  two  subsequent  2-hour  periods  the  rate  of  carbon- 
dioxid  emission  gave  even  sUghtly  higher  values  and  the  leaf  showed 
unmistakable  signs  of  injury,  so  that  these  results  are  not  included. 
In  the  experiment  described  under  1,  the  longer  periods  of  illumina- 
tion are  followed  by  an  increase  in  respiratory  activity.  It  is 
apparent  that  in  the  present  experiment,  however,  there  apparently 
has  not  been  sufficient  carbohydrate  synthesis  during  the  periods  of 
illumination  to  produce  an  increase  in  respiration  when  the  leaf  is 
again  put  in  the  dark. 

3.  In  the  two  preceding  experiments  the  concentration  of  carbon 
dioxid  was  relatively  low,  that  of  atmospheric  air.  In  the  following 
experiment  this  was  increased  ninefold.     The  leaf  was  cut  from  the 


96 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


plant  after  the  latter  had  been  exposed  to  the  sunlight  in  the  green- 
house for  7  hours.  The  respiration  and  photosynthesis  rates  were 
then  determined.  The  leaf  remained  in  the  frame  in  the  dark  with 
a  constant  air-stream  passing  over  it.  After  about  24  hours  the 
respiration  and  photosynthesis  rates  were  again  determined,  and 
likewise  after  about  48  hours.  The  results  of  this  experiment  are 
given  in  table  56  and  in  figure  26. 


Figure  26. 
Rates  of  photosynthesis  and 
respiration,  on  three  successive 
days,  of  a  leaf  of  Helianthus 
annuus  kept  in  the  dark.  The 
values  above  the  zero  line  indi- 
cate rates  of  photosynthesis, 
those  below,  rates  of  respira- 
tion, expressed  on  the  ordinates 
in  mg.  CO 2  fixed,  or  emitted, 
per  sq.  cm.  per  hour.  Data  and 
conditions  as  per  table  56. 


In  this  experiment,  in  which  the  carbon-dioxid  content  of  the  air 
was  greatly  increased,  the  rate  of  photosynthesis  shows,  as  in  the  pre- 
vious experiments,  a  course  which  in  general  parallels  that  of  the  res- 
piratory activity  of  the  leaf.  In  all  of  these  experiments  the  same 
source  and  intensity  of  illumination  was  employed. 

4.  The  carbon-dioxid  content  of  the  air-stream  was  increased 
further,  so  that  it  was  15.5  times  that  of  atmospheric  air.  In  this 
experiment  a  sunflower  leaf  was  also  used.  It  is  doubtful  whether 
with  the  intensity  of  Ught  employed  the  carbon  dioxid  was  the  limiting 
factor,  because  the  photosynthesis  per  square  centimeter  per  hour 
in  this  experiment  is  slightly  less  than  in  the  previous  one,  in  which 
the  carbon-dioxid  concentration  was  almost  half  of  that  used  in  the 
present  experiment.  In  table  57  and  figure  27  the  results  of  this 
experiment  are  given.  The  general  decline  of  the  rate  of  photosyn- 
thesis, with  decreasing  respiratory  activity,  also  holds  for  these 
conditions.     The  photosynthetic  rates  are  calculated  from  the  last 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


97 


preceding  respiration  rate.  For  this  reason  the  last  photosynthesis 
period,  No.  7,  appears  low.  Calculated  on  the  basis  of  the  next 
following  respiration  rate,  which  represents  more  nearly  the  true 
condition,  the  photosynthesis  rate  would  be  0.0523  instead  of  0.0413 
mg.  CO2  per  square  centimeter  per  hour. 

Table  57. — Rates  of  respiration  and  photosynthesis  of  a  leaf  of  Helianthus  anyiuus. 
The  CO2  concentration  was  15.5  times  that  of  atmospheric  air,  temperature  24°;   light  1,740 
lux;  area  of  leaf,  120.81  sq.  cm.    The  air-stream  equals  9.98  mg.  CO2  per  hour. 


No. 

Leaf 
ex- 
posed 
to- 

Time. 

Hrs. 

Total 
hours. 

Mg. 
CO2 
ab- 
sorbed. 

Mg. 

CO2 

ab- 
sorbed 

per 
hour. 

Respira- 
tion, mg. 

CO2 
emitted. 

Photo- 
synthesis, 
mg.  CO2 
fixed. 

Respira- 
tion per 

sq.  cm. 
per  hour 

in  mg. 
CO2. 

Photo- 
synthesis 

per  sq. 

cm.  per 

hour  in 
mg.  CO2 

2 
3 

4 
5 
6 

7 
8 

Dark 
Light 
Dark 

Dark 

Light 
Light 
Light 
Dark 

March  30. 

4  p.m.  to  5''30"  p.m 

3''45°'p.m.  to  7''15'°p.m.  . 
7''30™p.m.  to  9  p.m 

March  31, 
10''40°'  a.m.  to  12»'10°»  p.m. 
12''25">  p.m.  to  l''55°'p.m. 
1''55™  p.m.  to  3''25"  p.m. . 
3''25'"  p.m.  to  5''25'"  p.m. . 
5''35'"  p.m.  to  7''35°  p.m.  . 

1.5 
1.5 
1.5 

1.5 
1.5 
1.5 
2.0 
2.0 

1.50 
3.00 
4.50 

19.66 
21.16 
22.66 
24.66 
26.66 

18.66 
8.81 
18.66 

16.91 

9.56 

9.21 

12.56 

22.81 

12.44 
5.87 
12.44 

11.27 
6.57 
6.14 
6.28 

11.40 

2.46 

0.0204 

6.57 

0.0543 

2.46 
1.29 

0.0204 
0.0106 

4.90 
5.13 
4.99 

0.0405 
0.0424 
0.0413 

1.32 

0.0109 

In  the  experiments  described  under  2,  3,  and  4,  the  decrease  in 
photosynthetic  activity  occurs  after  the  leaves  had  remained  in  the 
dark  for  more  or  less  prolonged  periods.  The  possibiUty  naturally 
suggests  itself  that  the  reduction  might  be  due  to  a  decrease  in  the 
chlorophyll  of  the  leaves,  caused  by  keeping  the  plants  in  the  dark. 
On  the  basis  of  the  investigations  of  Willstaetter  and  Stoll,»  it  is  highly 
improbable  that  this  is  the  case.  They  kept  leaves  in  the  dark  at 
34°  to  37°  for  48  hours,  and  although,  toward  the  end  of  this  period, 
there  were  evidences  of  protoplasmic  disturbances  (abnormally  high 
CO2  evolution),  the  determinations  of  the  chlorophyll  components 
at  the  end  of  the  period  gave  the  same  results  as  normal  leaves. 

It  is  doubtful  whether  there  exists  between  the  respiratory  and 
photosynthetic  activities  anything  like  a  fixed  or  quantitative  ratio. 
The  quotients  determined  by  Pelster,  after  all,  represent  rather  con- 
fined conditions.  Similarly,  we  found  that  with  atmospheric  air  the 
quotients  are  about  of  the  same  magnitude  as  those  found  by  Pel- 
ster. With  increasing  carbon-dioxid  content,  however,  the  quanti- 
tative relation  of  these  values  changes,  although  the  relative  magni- 
tude seems  to  indicate  an  interrelationship.  Moreover,  this  inter- 
relationship appears  to  be  of  a  more  intricate  nature  than  if  it  were 


Willstaetter, 
p.  36. 


R.,  and  A.  Stoll.     Untersuchungen  ueber  die  Assimilation  der  Kohlensaeure, 


98 


STUDIES  IN  PLANT  RESPIRATION  AND  PHOTOSYNTHESIS. 


simply  a  case  of  high  photosynthesis  supplying  carbohydrates  in 
such  large  amounts  that  the  leaf  is  at  all  times  well  supplied  and  thus 
burns  this  sugar  at  a  correspondingly  high  rate. 

The  experiments  on  the  relation  of  respiration  and  photosynthesis 
are  being  extended,  especially  with  a  view  of  determining  the  temper- 
ature coefficients  under  a  variety  of  conditions.  There  is  considerable 
evidence  for  believing  that  the  photosynthetic  process  is  of  a  dual 


Figure  27. 
Rates  of  photosynthesis  and  respira- 
tion, on  successive  days,  of  a  leaf  of  Hdi- 
anthus  annuus.  The  values  above  the 
zero  line  indicate  rates  of  photosynthesis, 
those  below,  rates  of  respiration,  ex- 
pressed on  the  ordinates  in  mg.  CO2 
fixed,  or  emitted,  per  sq.  cm.  per  hour. 
Data  and  conditions  as  per  table  57. 


UD 


n 


u 


nature  or  even  more  complex,  involving  the  general  principle  of 
coupled  reactions.  That  one  of  these,  or  one  group  of  these,  reactions 
is  photochemical  there  seems  to  be  little  doubt.  The  nature  of  the 
other  reaction  (Willstaetter's  enzymatic  reaction),  or  that  associated 
with  the  respiratory  activity,  is  still  very  obscure.  In  what  manner 
these  two  sets  of  reactions  complement  each  other  or  how  they  are 
coupled  is  one  of  the  most  vital  points  of  the  photosynthesis  problem. 
Some  speculations  hereon  were  given  in  the  introductory  discussion 
of  this  section.  However,  at  this  stage  of  development  the  only 
method  which  promises  any  real  advance  is  the  experimental  one. 
In  view  of  the  importance  and  the  insight  which  can  be  gained  from 
the  interpretation  of  temperature  coefficients  on  the  basis  of  recent 
physical-chemical  investigations,  an  extension  of  these  determinations 
seems  highly  desirable  and  offers  another  means  of  analyzing  the 
phenomenon  of  photosynthesis. 


* 


